WO2003098220A1 - Modulation of mitosis by enhancing the interaction of cdc24a and cyclinb/cdk1 - Google Patents

Abstract

The invention generally relates to compositions and methods for modulating entry into mitosis. In one aspect, the invention provides methods for treating or preventing a hyperproliferative disorder in a mammal by promoting the phosphorylation of Cdc25A or the dephosphorylation of cyclin B/Cdk1, or by enhancing the activity of at least one molecular component of the Cdc25A and cyclin B/Cdk1 regulatory interaction/feedback pathway. In another aspect, the invention provides compositions capable of promoting the pathway. The invention has a wide range of important applications including providing screens for compounds that enhance the activity of one or more of the molecular components in the pathway and providing methods for screening a patient population.

Description

MODULATION OF MITOSIS BY ENHANCING THE INTERACTION OF CDC24A AND

CYCLINEB/CDK1

FIELD OF THE INVENTION

The invention generally relates to compositions and methods for modulating regulation of the cell cycle and entry into mitosis. In one aspect, the invention provides methods for treating or preventing a hyperproliferative disorder in a mammal by modulating a specific cell cycle autoregulatory pathway. In another aspect, the invention provides compositions capable of modulating this pathway. The invention has a wide range of important applications including providing screens for compounds that modulating the activity of this pathway.

BACKGROUND OF THE INVENTION

One of the fundamental biological processes conserved throughout evolution is the ability of all eukaryotes to secure high fidelity of the genetic transmission. Genomic stability is ensured by a plethora of cell cycle checkpoints, surveillance pathways which ultimately inhibit cyclin-dependent kinases (CDKs) and/or the anaphase-promoting complex, key enzymes that orchestrate progression through the cell cycle. Checkpoints capable of arresting mammalian cells in Gl phase, thereby preventing replication of the damaged DNA, appear to be largely dependent on the ability of the p53 tumor suppressor to induce the CDK inhibitor p21^WAF1/CIPl/SDIl. However, transient inhibition of Cdk2, the essential activity required for Gl/S progression, has been reported also in cells with compromised function of p53, and fibroblasts isolated from mice with homo-eygously deleted p21 gene still respond by at least attenuated delay of Gl/S transition upon DNA damage.

US Patent No: 5,756,335 (Beach & Galaktionov) discloses the cloning of the human Cdc25A and Cdc25B and identifies their activity as endogenous tyrosine phosphatases and their involvement in the cell cycle in regulating the activation of Cdc2-kinase. This patent discloses that Cdc25 A and Cdc25B are part of a multigene family having endogenous tyrosine kinase activity and which activate cyclin B in the absence of Cdc2. The patent shows that Cdc25 levels do not change in Xenopus in either the meiotic maturation or early embryonic division cycles. It further shows that Cdc25 physically associates with Cdc2/cyclin B in a cell cycle dependent manner, that maximal association between Cdc25 and Cdc2/cyclin B occurs at the time of maximal kinase activity and that Cdc2 associated with Cdc25 is tyrosine dephosphorylated and active as a kinase. The patent discloses that Cdc25A is overexpressed in some forms of cancer and suggests regulating the activity of Cdc2 by controlling the interaction of Cdc25 with Cdc2, cyclin B or the Cdc2/cyclin B complex. Methods of treating hyperproliferative disorders such as cancer are suggested using anti-sense oligonucleotides to block Cdc25 production, blocking a Cdc25 transcription factor, degrading Cdc25 with a protease, using agents capable of binding to Cdc25 or cyclin B or inhibiting the activating domain of cyclin B.

WO99/11795 discloses the identification of human effector cell cycle checkpoint kinase 1 (Chkl) and that expression of Chkl decreases the sensitivity of the cell to DNA damage. The cloning of cell cycle checkpoint kinase 2 (Chk2) is described in Matsuoka et al, 1998. Further Chk2 sequences are disclosed in Chaturvedi et al, 1999, and Blasina et al, 1999a.

There have been attempts to understand how cells preserve genetic integrity. For example, it has been reported that mammalian cells exposed to ionizing radiation (IR) activate the ATM kinase, a recognized S-phase checkpoint. Defects in the ATM kinase as well as additional proteins such as Nbsl, Chk2 and Mrel 1 have been disclosed. There have been reports that genetic replication defects may be associated with inherited, cancer-prone diseases such as ataxia telangiectasia (A-T), Nijmegen breakage syndrome (NBS), and ataxia-telangiectasia-like disorder (ATLD). ATM-mediated phosphorylation of the Nbsl protein is thought to be important for Mrel 1 complex-dependent activation of the RDS checkpoint. ATM has also been reported to phosphorylate and activate the checkpoint signaling kinase Chk2. There has been no recognition that the ATM, Nbsl and Mrel 1 proteins associate to form a distinct DNA replication pathway.

In addition to the roles played by key enzymes at checkpoints in the Gl/S phase transition, there have been attempts to understand the roles played by key enzymes at checkpoints in other phase transitions, such as the G2/M transition. Likewise, there have been attempts to understand the molecular mechanisms underlying cellular multiplication and maintenance of genomic integrity, which require precise timing, velocity and spatial distribution of phosphorylation events carried out by CDKs. For example, CDKs are thought to be subject to complex regulation in response to signals from the extracellular environment or cell cycle checkpoints.

In mammals, the Cdc25 family includes three homologs, termed Cdc25A, B, and C. Cdc25B and C are regarded as mitotic regulators and may serve as targets of those checkpoint pathways that delay entry into mitosis in response to DNA damage or stalled replication. Despite the recent progress, the understanding of overlapping and unique roles of the Cdc25 phosphatases at various cell cycle transitions is incomplete.

It would be useful to have compositions and methods for modulating entry into mitosis, particularly a pathway that includes Cdc25 A. It would be particularly useful to have screens for detecting compounds that can increase or decrease activity of that pathway in vivo and in vitro.

SUMMARY OF THE INVENTION

Broadly, the present invention is based on the discovery of the role of Cdc25A in a further signaling pathway for entry into mitosis (i.e., promotion of the G2 M transition). In one signaling pathway, Chkl or Chk2 is activated following DNA damage and phosphorylates Cdc25 A at one or more serine residues. The phosphorylated Cdc25 A is then recognized by the F-box protein part of ubiquitin ligase and is then degraded in a proteasome dependent manner, thereby allowing the cells to undergo cell cycle arrest and repair. Accordingly, by interfering with the phosphorylation and/or degradation of Cdc25A and or using other strategies to maintain Cdc25A level, this pathway can be inhibited, preventing cells from undergoing repair and thereby increasing the accumulation of DNA damage in the cells. By directing this treatment so that the accumulation of DNA damage is favored in populations of diseased cells, it is in turn possible to increase the fraction of such cells which can be killed by DNA damaging therapeutic agents, such as radiation or anti-tumor drugs, or which undergo apoptosis. As tumor cells are often defective in other checkpoints (e.g. many tumor cells are p53 defective) and repair pathways as compared to normal cells, this means that the approach can be used to preferentially sensitize the tumor cells to these treatments, reducing the severity of side effects associated with these treatments or increasing their effectiveness.

DNA replication and repair is also modulated by a second pathway. That pathway is substantially independent of the Cdc25A signaling pathway and is generally referred to herein as a "ATM-Nbsl/Mrel 1 " DNA replication pathway or related phrase. The ATM-Nbsl/Mrel 1 pathway is distinct from the Cdc25A pathway and provides a further cell signaling route for reducing or blocking DNA replication in response to DNA damage. More particularly, it has been found that the ATM-Nbsl/Mrel 1 pathway involves cooperation between the ATM- kinase and Nbsl ; and between Nbsl and the Mrel 1 protein. Inhibition of the ATM- Nbsl/Mrel 1 pathway in accord with this invention further provides for enhanced DNA damage in response to radiation, chemical agents, anti-tumor drugs and the like.

The present invention is grounded in the discovery of a third pathway involving Cdc25A in the regulation of the cell cycle. It has been discovered that Cdc25A stability undergoes dramatic changes at the G2/M transition. Thus, Cdc25A becomes abruptly stabilized upon entry into mitosis and contributes to the cellular phosphatase pool required to dephosphorylate Cdkl fully. In addition, DNA damage-induced inhibition of mitotic entry is accompoanied by destruction of Cdc25A, an event required for the productive G2/M arrest. These novel functions and regulatory modes of Cdc25A highlight a model of phosphorylation- mediated switches among multiple, differentially stable states of a cell cycle-regulatory protein as a flexible way to re-set its activity thresholds under diverse biological conditions. In essence, there are at least three states of phosphorylation for Cdc25A: a non-phosphorylated, labile state; a phosphorylated, ultra-labile state, which is rapidly degraded by the proteasome; and a differentially phosphorylated, stable state.

The invention generally relates to compositions and methods for modulating regulation of the cell cycle and entry into mitosis. The invention provides methods for treating or preventing a hyperproliferative disorder in a mammal by modulating the Cdc25A-cyclin B/Cdkl autoregulatory pathway, such as by providing compositions and compositions capable of modulating this pathway. In addition, the invention has a wide range of important applications including providing screens for compounds that modulate the activity of this pathway. "Modulating" includes the use of compounds or methods that can increase or decrease activity in vivo and in vitro. Modulating entry into mitosis can be achieved by modulating the activity of the Cdc25A-cyclin B/Cdkl pathway, such as by modulating the amount of Cdc25A, particularly its stable form, or the amount of cyclin B/Cdkl, particularly its active form. Modulating the activity of the pathway can also include increasing or decreasing the presence or activity of inhibitors or enhancers/activators of Cdc2 A, cyclin B, Cdkl, or a combination thereof.

It is believed that the invention is better appreciated by reference to Figure 6.

Accordingly, and in one aspect, the invention provides a method for treating or preventing cancer or a hyperproliferative disorder comprising administering a composition which is capable of enhancing the interaction of Cdc25 A and cyclinB/Cdkl .

In a further aspect, the invention provides a composition having the property of promoting the phosphorylation of Cdc25A or a derivative thereof, wherein the Cdc25A or the derivative thereof, includes a serine residue at a position corresponding to amino acid Ser 17 or Serl 15 in Cdc25A, and wherein the composition comprises a protein complex comprising:

(a) a first protein moiety comprising:

(i) a protein which has at least 80% sequence identity with a corresponding sequence of cyclin B; or

(ii) a derivative of protein (ai); or

(iii) a substance which is protein (ai) or derivative (aii) linked to a coupling partner; and

(b) a second protein moiety comprising:

(i) a protein which has at least 80% sequence identity with a corresponding sequence of Cdkl; or

(ii) a derivative of protein (bi); or

(iii) a substance which is protein (bi) or derivative (bii) linked to a coupling partner.

In a further aspect, the invention provides a composition having the property of enhancing the activity of cyclin B/Cdkl and promoting the phosphorylation of Cdc25A by the cyclin B/Cdkl, the composition comprising:

(a) a protein which has at least 80% sequence identity with a corresponding sequence of Cdc25A, the protein including a serine residue at a position corresponding to amino acid Serl7 or Serl 15 in Cdc25A; or,

(b) a derivative of protein (a); or

(c) a substance which is protein (a) or derivative (b) linked to a coupling partner.

In yet another aspect, the invention provides a method of identifying compounds capable of modulating the interaction of Cdc25A and cyclin B/Cdkl kinase, the method comprising:

(a) contacting (i) a composition comprising Cdc25A or a fragment or variant thereof, (ii) a composition comprising cyclin B/Cdkl kinase or a fragment or variant thereof and (iii) a candidate compound, under conditions wherein, in the absence of the candidate compound, the compositions interact; and

(b) determining the interaction between the compositions to identify whether the candidate compound modulates the interaction.

In another aspect, the invention provides a method of identifying binding partners of a composition having the property of enhancing the activity of cyclin B/Cdkl kinase and promoting the phosphorylation of Cdc25A by the cyclin B/Cdkl kinase, the composition comprising a protein having at least 80% sequence identity with a corresponding sequence of Cdc25A, the protein including serine at a position corresponding to amino acid Serl7 or Serl 15 in Cdc25A, the method comprising contacting the composition and a candidate compound and determining whether the candidate compound has the property of binding to the composition.

In yet another aspect, the invention provides a compound comprising an amino acid motif having between 2 and 30 amino acids from Cdc25A and having a serine at a position corresponding to Serl 7 or Serl 15 in full length Cdc25A in the design of an compound which is modelled to resemble the three dimensional structure, the steric size, and/or the charge distribution of the amino acid motif, the wherein the compound has the property of binding to cyclin B/Cdkl.

In yet another aspect, the invention provides a method of preparing a medicament for the treatment of cancer or a hyperproliferative disorder comprising combining a composition which is capable of enhancing the interaction of Cdc25A and cyclin B/Cdkl, wherein the enhancement of the interaction promotes the phosphorylation of the Cdc25A on a serine at a position corresponding to Serl7 or Serl 15 in a full length Cdc25A, with a pharmaceutically acceptable carrier.

In yet a further aspect, the invention provides a method for raising antibodies capable of specifically binding to phosphorylated Cdc25A, comprising use of a composition which is:

(a) a peptide fragment of between 5 and 30 amino acids which has at least 80% sequence identity -with a corresponding sequence of Cdc25 A, the fragment including a phosphorylated serine residue at a position corresponding to amino acid Serl 7 or Serl 15 in Cdc25A; or,

(b) a derivative of peptide fragment (a); or

(c) a substance which is peptide fragment (a) or derivative (b) linked to an immunogenic carrier.

In another aspect, the invention provides a method for the preparation of a medicament for the treatment of cancer or a hyperproliferative disorder, comprising combining a composition which is capable of enhancing the interaction of cyclin B/Cdkl kinase and Cdc25A, wherein the enhancement of the interaction promotes the phosphorylation of the Cdc25A, with a pharmaceutically acceptable carrier.

In yet another aspect, the invention provides a method of identification of patients having a functional Cdc25A phosphorylation-cyclin B/Cdkl kinase pathway in cancer cells comprising measuring the presence or absence of Cdc25A phosphorylation on Serl 7 or Serl 15 following treatment of the cells with chemotherapy or radiotherapy.

In yet another aspect, the invention provides a diagnostic kit for the identification of patients expressing Cdc25A, wherein the Cdc25A is phosphorylated on Serl7 or Serl 15 in cancer cells comprising an antibody against phosphorylated Cdc25A.

In a further aspect, the invention provides a method of screening a patient population by determining the level of Cdc25A phosphorylation in cancer cells derived from each patient comprising:

(a) providing a sample comprising tissue material or cells from a tumor;

(b) preparing the sample to obtain an appropriate tissue extract;

(c) optionally further treating the sample extract by one or more purification processes;

(d) contacting the tissue preparation with an antibody against phosphorylated Cdc25A to produce a Cdc25A-antibody primary complex;

(e) contacting the complex with a secondary antibody containing a specific label or reporter group to enable determination of the amount of Cdc25A present; and

(f) either

(i) comparing the amount of Cdc25 A with a standard sample to determine whether it is more or less than the standard amount; or (ii) comparing the level of activity of Cdc25 A with that of wild-type ^' Cdc25A, wherein the wild-type Cdc25A has been phosphorylated on Serl 7 or Serl 15 or both, in a standard protein activity assay to determine whether it is more or less than the standard amount.

In another aspect, the invention provides a method of screening a patient population by determining the presence of phosphorylated Cdc25A in cancer cells derived from each patient comprising:

(a) providing a sample comprising tissue material or cells from a tumor;

(b) preparing the sample to obtain an appropriate tissue extract under conditions selected to inhibit phosphorylation and dephosphorylation;

(c) contacting the tissue preparation with an antibody against Cdc25A to produce a Cdc5A-antibody complex;

(d) isolating the Cdc25A under conditions selected to inhibit phosphorylation or dephosphorylation;

(e) simultaneously subjecting to a size separation method under conditions selected to inhibit phosphorylation or dephosphorylation:

(i) the Cdc25A;

(ii) at least one Cdc25A standard comprising phosphorylated or non- phosphorylated Cdc25A;

(f) detecting the presence of the Cdc25A and the Cdc25A standard; and

(g) observing the relative migration of the Cdc25A as compared to the Cdc25A standard, whereby phosphorylated Cdc25A migrates more slowly than non- phosphorylated Cdc24A.

In another aspect, the invention provides a method of sensitizing a patient for chemotherapy or radiotherapy or antisense therapy comprising administering at least one drug capable of promoting the Cdc25A-cyclin B/Cdkl pathway.

Embodiments of the present invention will now be described by way of example with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE DRAWINGS

Figures 1A to IH depict mitotic stabilisation of Cdc25A.

Figure 1A is a series of photographs of gels, accompanied by graphs, providing experimental results showing that Cdc25A is rapidly turned over throughout the interphase and stabilised in mitosis. U-2-OS cells were released from a double thymidine block for 4, 9, or 18 h to obtain cells in S, G2, or Gl phase, respectively, or arrested in prometaphase (M) by treatment with Nocodazole.

Figure IB is a series of photographs of a gel providing experimental results showing differential stability of Cdc25A in asynchronous (AS) vs. mitotic (M, purified by shake-off after nocodazole-treatment) cells, compared with Cdc25C control, in an extended cycloheximid experiment analogous to A.

Figure 1C is a series of photographs showing Cdc25A protein turnover measured in exponentially growing U-2-OS cells by a pulse-chase of [³⁵S]-methionine-labeled protein lysate followed by immunopreciptation and autoradiography (top), and cycloheximide treatment followed by Western blotting (bottom), respectively.

Figure ID is a series of photographs showing the abundance of Cdc25A, Cdc25C, and a control hPBGD mRNA, determined by RT-PCR of poly-A+ RNA isolated from asynchronous or mitotic cells.

Figure IE is a photograph of a Western blot showing that interphase but not mitotic Cdc25 A accumulates after inhibition of the proteasome.

Figure IF is a series of photographs of a Western blot showing that Cdc25A is hyperphosphorylated in mitosis independent of Nocodazole-treatment. Mitotic cells were obtained by shake-off after Nocodazole (+), or upon release for 14 h from a double-thymidine block (-). Cdc25A was analysed by Western blotting.

Figure IG is a series of photographs showing that M-phase-specific phosphorylation regulates Cdc25C but not Cdc25A activity. Cdc25 proteins were immunoprecipitated from asynchronous or mitotic cells, left untreated or dephosphorylated with λ-phosphatase (PPase) and assayed for Cdc25 phosphatase activity.

Figure IH is a series of blotting photographs showing that Cdc25A is destabilised after release from metaphase arrest. Cells were released from a Nocodazole-block by replating into a drug-free medium. At the indicated times, cell lysates were processed for Western blotting or cyclin B/Cdkl activity measurement.

Figures 2A to 2C show cyclin B/Cdkl -targeted phosphorylation sites of Cdc25A.

Figure 2A is a series of photographs showing that cyclin B/Cdkl phosphorylates Cdc25A in vitro. Cyclin B/Cdkl immunoprecipitated (IP) from asynchronous or nocodazole-arrested cells was assayed using full-length GST-Cdc25A as substrate; Ros=Roscovitine; WB=Western blot;

CdklpTyr=inactive, Tyrl5-phoshorylated Cdkl.

Figure 2B is a series of Western blot photographs showing that inhibition of cyclin B/Cdkl destabilises Cdc25A in mitosis by measuring Cdc25A and Cdc 27 levels.

Figure 2C is a combination schematic and sequence comparison depicting conservation of

Serl7 and Serl 15 of Cdc25A in different species.

Figures 3A to 3F show that phosphorylation by cyclin B/Cdkl uncouples Cdc25A from ubiquitylation and degradation.

Figures 3 A and 3B are photographs showing the results of assays demonstrating that cyclin

B/Cdkl phosphorylates Cdc25A on Serl7 and Serl 15 in vitro. Cyclin B/Cdkl was immunoprecipitated from mitotic U-2-OS cells and assayed using the indicated GST-tagged fragments of Cdc25A containing Serl 7 or Serl 15 or Alanine substitutions as substrates, with or without Roscovitine (Ros).

Figure 3C is a series of Western blot photographs. U-2-OS T-Rex cells were transfected with plasmids encoding wildtype Cdc25A or the S17/S115 double mutant (A/A), treated with

Nocodazole or left asynchronous (AS), and induced by addition of tetracycline for 3 or 6h.

When indicated, LLnL was added to the medium at the time of the transgene induction.

Cdc25A proteins were analysed by Western blotting.

Figure 3D is a Western blot photograph showoing destabilisation of Cdc25 A(A/A) in mitosis.

U-2-OS T-Rex cells were treated as in C, followed by addition of CHX for 2h. The level of

Cdc25A was assessed by Western blotting.

Figure 3E is a photograph of experimental results of in vivo assays showing that Cdc25A is not ubiquitylated in mitosis. Asynchronous (AS) or mitotic (M) U-2-OS/B3C4 cells transiently transfected with His-ubiquitin were kept uninduced or induced to express ectopic Cdc25A for 12 h as indicated, and processed for detection of Cdc25A-associated ubiquitn (Ub) conjugates. Figure 3F is a photograph of experimental results of in vivo assays showing that mutation of the cyclin B/Cdkl -targeted sites in Cdc25A restores its ubiquitylation in mitosis. U-2- OS/Cdc25A(A/A) cells were treated and analysed as in D or E.

Figures 4A to 4H show that Cdc25A activates cyclin B/Cdkl and promotes mitotic entry. Figure 4A is a series of photographs showing Western blot-verified depletion of Cdc25A, B and C from U-2-OS cells lysed in a kinase buffer. Individual and/or the indicated combinations of Cdc25 proteins were depleted by specific antibodies or control preimmune (Preimm.) serum via in vitro assays.

Figure 4B is a photograph of results of in vitro assays showing that Cdc25 isoforms contribute to activation of Cyclin B/Cdkl. Cell lysates were prepared and depleted as in A, supplemented with 10 mM EDTA to inhibit endogenous kinases and incubated for 20 min at 30°C. Cyclin B/Cdkl was then immunoprecipitated (IP) and its activity measured using Histone HI as a substrate. Sodium vanadate (+Van) was added into the control reaction to inhibit all Cdc25 phosphatase activity. Numbers indicate the extent (%) of cyclin B/Cdkl activation relative to the control depletion with preimmune serum. (*)Lysate was not incubated at 30°C. Figure 4C is a series of Western blot photographs showing that Cdc25A binds cyclin B/Cdkl and stimulates its activity. U-2-OS/B3C4 cells were induced (-Tet) to express Cdc25A for 12 h, and cyclin BI and Cdc25A immunoprecipitated as indicated. Cyclin BI immunoprecipitates were assayed for histone HI kinase activity; Cdc25A immunocomplexes were analysed for associated cyclin BI and Cdkl by Western blotting.

Figure 4D is a pair of graphs providing the results of a flow cytometry analysis showing that elevated Cdc25A induces premature mitotic entry. U-2-OS/B3C4 cells were arrested in S phase by a double thymidine block, released and induced to express ectopic Cdc25A (-Tet) or kept uninduced (+Tet). Five hours later, cells were analysed by flow cytometry for phospho- Histone H3, a marker of productive entry into mitosis.

Figure 4E is a series of photographs showing that loss of specific phosphatase activity in U-2- OS/Cdc25A PD cells conditionally expressing a phosphatase-dead (PD) allele of Cdc25A. The indicated cell lines were induced to express wild-type (WT) and phosphatase-dead (PD) Cdc25A, respectively, for 24 hours and assayed for Cdc25A-associate phosphatase activity. The parental U-2-OS/TA cell line was used as a control to determine the non-specific background activity associated with anti-HA antibody. Figure 4F is a series of graphs providing the results of a flow cytometry analysis showing that dominant-negative Cdc25A delays entry into S phase. U-2-OS/Cdc25A PD cells were arrested in mitosis with Nocodazole, induced to express Cdc25A PD (as in E), and then released to re- enter Gl and analysed for DNA content by flow cytometry 14 h later. Figure 4G is a graph providing the results of a flow cytometry analysis showing that dominant-negative Cdc25A interferes with G2/M transition. U-2-OS/Cdc25A PD cells were arrested by double thymidine block, released and induced to express Cdc25A PD (-Tet) or kept uninduced (+Tet). At the indicate times, DNA content was analysed by flow cytometry. Figure 4H is a series of photographs showing that overexpression of the phosphatase-dead Cdc25A does not impair the activity of endogenous Cdc25B and Cdc25C. U-2-OS/Cdc25A PD cells were induced for 24 hours and assayed for the activity associated with the indicated Cdc25 phosphatases.

Figures 5A to 5C show Cdc25A degradation in response to DNA damage in G2.

Figure 5A is a series of photographs showing that etoposide-induced G2/M arrest causes loss of Cdc25A protein and associated activity. Asynchronous U-2-OS cells were treated with

Etoposide for 12 h to arrest cells in G2, and levels and activities of Cdc25A and C were determined.

Figure 5B is a series of graphs providing the results of a flow cytometry analysis showing that overexpression of Cdc25A but not Cdc25C compromises the G2/M checkpoint. Etoposide was added to asynchronous Cdc25A- and Cdc25C-inducible, or parental U-2-OS/TA cells. Four hours later, Nocodazole was added to the medium and cells were concomitantly induced to express the transgenes. After additional 16 hours, cells were analysed by flow cytometry for phospho-histone H3,

Figure 5C is a Western blot photograph showing that the mitotic form of Cdc25 A is not destabilised by DNA damage. Asynchronous or mitotic U-2-OS cells were γ-irradiated (10

Gy), and Cdc25A analysed by Western blotting 1 hour later.

Figures 6A to 6B depict models of Cdc25A regulation and function in the mammalian cell cycle.

Figure 6 A is a schematic depicting the model of a dual positive feedback loop leading to maximal and irreversible activation of the M-phase-promoting cyclin B/Cdkl kinase. See

Summary and Detailed Description. Figure 6B is a chart depicting the model of the phosphorylation-dependent switches among three differentially stable forms of Cdc25A, designated: labile, ultra-labile, and stable, which determine thresholds and roles of Cdc25A in unperturbed or DNA-damaged interphase, and in mitotic cells, respectively. See Summary and Detailed Description.

DETAILED DESCRIPTION

The Cdc25 A degradation pathway in response to DNA damage operates to transiently delay cell cycle progression in both Gl phase and inside S phase, to provide time for DNA repair and to increase cell survival. During studies of protein turnover of human Cdc25 phosphatases, it was found that Cdc25A stability undergoes dramatic changes at the G2/M transition. Thus, Cdc25A became abruptly stabiKsed upon entry into mitosis and contributed to the cellular phosphatase pool required to fully dephosphorylate Cdkl. In addition, DNA damage-induced inhibition of mitotic entry was accompanied by destruction of Cdc25A, an event required for the productive G2/M arrest. These results point to novel functions and regulatory modes of Cdc25A, and highlight a concept of phosphorylation-mediated switches among multiple, differentially stable states of a cell cycle-regulatory protein as a flexible way to re-set its activity thresholds under diverse biological conditions.

The Cell Cycle

The cell cycle checkpoint pathways collectively represent an important part of cell cycle control which do not interfere with normal cell proliferation, but rather monitor the progression through the cell cycle in terms of the quality of DNA, precision of DNA replication and chromosome segregation. In other words, checkpoints are a quality and fidelity control, monitoring the performance of the basic cell cycle machinery with the option to stop the cell cycle in the event DNA becomes damaged, DNA replication machinery makes errors or chromosomes are not ready to be separated properly. The cell cycle checkpoint mechanisms which are activated in response to DNA damage therefore provide time for DNA repair, and sometimes also help activate repair, allowing cells to survive such damage (if the damage is reparable), or to prevent cell cycle progression in cells with unrepaired DNA or abnormal chromosomes.

Defects or prevention of the cell cycle checkpoint functions result in the accumulation of unrepaired genetic damage or mutations, and that cells in which one or more checlφoints do not function properly are generally more sensitive to DNA damage and die more readily when exposed to radiation or other genotoxic agents than normal cells. Unrepaired or excessive DNA damage eventually triggers the cell suicide mechanisms of apoptosis or mitotic catastrophe, thus eliminating the potentially dangerous, genetically highly unstable cells. Probably the most significant difference between normal and tumor cells is that most normal cells are non-proliferating, and even those which do proliferate harbor multiple functional checkpoint pathways which can respond to DNA damage to minimize genetic destabilization (mutations). In contrast, tumor cells proliferate, lack one or several checkpoint control pathways and sometimes are also defective in the DNA repair, all features which make them more susceptible to DNA damage. In retrospect, it appears that many anti-cancer therapeutics originally found empirically, such as γ-radiation and cytotoxic drugs, operate via inducing DNA damage and that these agents are successful in view of the differences between normal and cancer cells mentioned above.

The issue of selectivity for tumors relies, at least in part, on the fact that most tumors lack some checkpoints already, unlike normal cells which can use the whole spectrum of checkpoints to reversibly block the cell cycle, and later recover. Tumor cells, meanwhile, might for instance keep the Cdc25A pathway as the only (or one of two, perhaps) remaining active checkpoint, and its neutralization by our future strategy would deprive tumor cells even of this remaining safeguard mechanism against excessive DNA damage and cell death.

Thus, one strategy is to interfere with a function of a particular checkpoint mechanism to treat cancer or a hyperproliferative disorder in which the checkpoint pathway is operational, while simultaneously or sequentially treating a patient with conventional chemotherapy or radiotherapy. The aim of this strategy is to deprive the diseased cells of the possibility to implement the checkpoint and so deny the cells the time for proper repair. Deprived of this possibility, the affected cells shift the balance of their decisions-reactions towards irreversible arrest or cell death. The induction of cell death is more likely to occur in tumor cells than in normal cells, since the latter have multiple checkpoints operational, have more efficient repair, or proliferate less, and as a result are much more likely to survive and properly repair the effects of the treatment.

The rapid phosphorylation and degradation of Cdc25A blocking cells at Gl/S and in S phase belongs to the 'classical' reversible category. Thus, the degradation and or inactivation of Cdc25A following DNA damage is rapid and only temporary (imposed within 30-60 minutes and lasting for some 3-4 hours when rather low doses of UV or other DNA damaging agents are used), and the majority of the cells do recover from this transient arrest and continue proliferating.

Cdc25 A is required as a positive cell cycle regulator in all somatic cycling cells, due to its ability to remove the inhibitory phosphate from cyclin-dependent kinases such as cdk2, the partner of cyclin E and cyclin A at the Gl/S transition and in S phase.

Cdc25A serves as a target of the novel checkpoint mechanism activated in response to DNA damage in which the Cdc25A protein becomes phosphorylated and degraded, thereby preventing its essential positive role for Gl/S and resulting in cell cycle arrest. This pathway is only operational when cells are exposed to DNA damage.

Thus, interrupting the signaling pathway to Cdc25A after DNA damage or interfering with execution of this pathway human tumor cells, thereby increases the accumulation of DNA damage and the fraction of cells normally killed by the DNA damaging therapeutics such as radiation and anti-tumor drugs. The aim of this strategy is therefore to sensitize tumor cells towards the action of the DNA-damaging cancer treatments with the scope to either achieve the same treatment effects with lower doses of radiation/drugs, thereby decreasing some of the adverse side effects of the existing therapies, or achieving more pronounced elimination of diseased cells.

The residue of Cdc25 A targeted for phosphorylation by Chkl or Chk2 appears to be a serine residues and more particularly Serl23 or Ser262 or Ser292 or Ser504 of the human Cdc25 A, as deduced from phosphopeptide mapping of Cdc25 A isolated from cells after DNA damage, and confirmed by site-directed mutagenesis. The phosphosite corresponding to Ser292 is a dominant one upon response to both UV or gamma radiation, i.e. the site is shared by Chkl and Chk2. Thus, when disrupting peptides are delivered into cells, either by microinjection, electroporation or by use of a coupling partner such as a transport molecule which allow the peptides to cross cell membranes, just before or concomitant with DNA damage insults such as radiation, they can compete for binding to activated Chkl or Chk2 kinases in the cells, and thereby inhibit or even prevent the interaction of the kinase with the endogenous Cdc25A, in turn resulting in the presence of an amount of Cdc25A sufficient to inhibit or prevent the cells employing the checkpoint.

Alternatively, the downstream recognition of the phosphorylated Cdc25A protein by a protein ubiquitination enzyme, such as a member of the "F-box protein" family of proteins, which are components of ubiquitin ligases recognizing the specific phosphorylated residue in proteins targeted for ubiquitination and degradation, can be targeted to inhibit or prevent it leading to reduction of Cdc25 A levels in response to DNA damage. Checkpoint pathways in eukaryotic cells are activated when the cells are exposed to ionizing radiation (TR) in order to delay cell cycle progression. Defects in the IR-induced S-phase checkpoint cause radioresistant DNA synthesis (RDS), a phenomenon identified in cancer-prone ataxia- telangiectasia (A-T) patients suffering mutations in the ATM gene. There is an interplay mechanism between ATM, Chk2 kinase and Cdc25A phosphatase, and deregulation of this mechanism leads to RDS. Cdc25A activates cyclin dependent kinase 2 (Cdk2) required for DNA replication, and it becomes degraded in response to DNA damage. IR-induced destruction of Cdc25A requires ATM and Chk2-mediated phosphorylation on serine 123. The resulting loss of Cdc25A protein precludes activating dephosphorylation of Cdk2 and leads to transient DNA replication blockade. The ATM-Chk2-Cdc25A-Cdk2 pathway serves as a genomic integrity checkpoint, which prevents RDS.

The present invention is grounded in the discovery of a third pathway involving Cdc25A in the regulation of the cell cycle. It has been discovered that Cdc25A stability undergoes dramatic changes at the G2/M transition. Thus, Cdc25A becomes abruptly stabilized upon entry into mitosis and contributes to the cellular phosphatase pool required to dephosphorylate Cdkl fully. In addition, DNA damage-induced inhibition of mitotic entry is accompoanied by destruction of Cdc25 A, an event required for the productive G2/M arrest. These novel functions and regulatory modes of Cdc25A highlight a model of phosphorylation- mediated switches among multiple, differentially stable states of a cell cycle-regulatory protein as a flexible way to re-set its activity thresholds under diverse biological conditions. In essence, there are at least three states of phosphorylation for Cdc25A: a non-phosphorylated, labile state; a phosphorylated, ultra-labile state, which is rapidly degraded by the proteasome; and a differentially phosphorylated, stable state. Peptides and Peptido imetics

The wild type Cdc25A amino acid and nucleic acid sequences are disclosed in US Patent No:5,441,880 and WO93/10242. The sequence annotation for the amino acid residues of Cdc25A used herein are in accordance with the annotation used in US Patent 5,441,880. However, the NCBI protein database accession XP_037169 submitted 23-AUG-2001 has a slightly different sequence of the first 13 amino acid residues of the human Cdc25A resulting in the serine residues 123, 262, 292 and 504 being annotated as serine 124, 263, 293 and 505. The invention utilizes proteins, derivatives of proteins (including peptides and peptide fragments), and compositions which are proteins or derivatives of proteins linked to a coupling partner.

Preferably, the peptide is based on the Cdc25A sequence and includes the phosphorylation sites targeted by cyclinB/Cdkl, namely Serl7 and/or Serl 15 of wild type human Cdc25A, e.g. comprising a motif of 2, 3, 4, 5, 6, 7, 8, 10, 20 or 30 amino acids from the sequence below and including a serine residue in a position corresponding to Serl7 or Serl 15 of Cdc25A, optionally in combination with one or more amino acid alterations as discussed below. The sequences surrounding the Serl7 and Serl 15 phosphorylation sites in human Cdc25A are shown in Figure 2C, along with the corresponding sequences in mouse and rat. These regions are highly conserved in mammals.

In other embodiments, the present invention provides variant peptides based on this motif of Cdc25A in which Serl7 or Serl 15 is substituted by a residue such as alanine which is non-phosphorylatable or resists phosphorylation, or by a residue such glutamic acid which mimics phosphorylation.

In other embodiments, the present invention provides variant peptides in which the serine residue is phosphorylated. These peptides may be used along with any of the peptides disclosed herein to immunize mice and rabbits to prepare antibodies specifically recognizing the Cdc25A, and more especially antibodies which are capable of specifically binding to Cdc25A which has been phosphorylated by cyclin B/Cdkl or which is unphosphorylated. The phosphorlyated Cdc25A peptides, and especially those phosphorylated at Serl7 or Serl 15, can also be used, possibly in a cell permeable form, to try to maintain the level of phosphorylated Cdc25A or to maintain or enhance the activation of the cyclin B/Cdkl kinase. This method could be employed alone or in combination with other approaches described herein.

As is well understood, identity at the amino acid level is generally defined and determined by the TBLASTN program, of Altschul et al, J. Mol. Biol., 215:403-10, 1990, which is in standard use in the art. Sequence identity may be over the full-length of the relevant peptide or over a contiguous sequence of about 5, 10, 15, 20, 25, 30 or 35 amino acids, compared with the relevant wild-type amino acid sequence. Preferably, the amino acid sequence of the peptides of the invention share at least 75%, or 80%, or 85% identity, and more preferably at least 90% or 95% identity sequence identity with the corresponding part of the full length human Cdc25A, cyclin B, or Cdkl sequences.

The present invention also provides sequence variants of the above peptides. In one embodiment, the variants are peptide fragments of Cdc25A including 1, 2, 3, 4, 5, greater than 5, or greater than 10 amino acid alterations such as substitutions, deletions or insertions with respect to the wild-type sequence.

Peptide or protein derivatives of the peptides or proteins and sequence variants described above include pharmaceutically acceptable salts of the peptides or proteins, alkyl esters, amides, alkylamides, dialkylamides, wherein the alkyl groups are preferably lower alkyl such as Cl-4.

The present invention further includes provides peptides or proteins which are composed of D and L amino acids, or combinations thereof. Alternatively or additionally, the proteins, peptides, variants and derivatives may be part of a larger peptide, which may or may not include an additional portion of Cdc25A, e.g. 1, 2, 3, 4, 5 or 10 or more additional amino acids, adjacent to the relevant specific peptide fragment in Cdc25A, or heterologous thereto may be included at one end or both ends of the protein or peptide.

Coupling partners

The invention also includes derivatives of the peptides, including the peptide linked to a coupling partner, e.g. an effector molecule, an immunogen, a label, a drug, a toxin and/or a carrier or transport molecule. Techniques for coupling the peptides of the invention to both peptidyl and non-peptidyl coupling partners are well known in the art. In one embodiment, the carrier molecule is a 16 aa peptide sequence derived from the homeodomain of Antennapedia (e.g. as sold under the name "Penetratin"), which can be coupled to a peptide via a terminal Cys residue. The "Penetratin" molecule and its properties are described in WO91/18981.

Synthesis

Peptides may be generated wholly or partly by chemical synthesis. The compounds of the present invention can be readily prepared according to well-established, standard liquid or, preferably, solid-phase peptide synthesis methods, general descriptions of which are broadly available (see, for example, in J.M. Stewart and J.D. Young, Solid Phase Peptide Synthesis, 2nd edition, Pierce Chemical Company, Rockford, Illinois (1984), in M. Bodanzsky and A. Bodanzsky, The Practice of Peptide Synthesis, Springer Verlag, New York (1984); and Applied Biosystems 430A Users Manual, ABI Inc., Foster City, California), or they may be prepared in solution, by the liquid phase method or by any combination of solid-phase, liquid phase and solution chemistry, e.g. by first completing the respective peptide portion and then, if desired and appropriate, after removal of any protecting groups being present, by introduction of the residue X by reaction of the respective carbonic or sulfonic acid or a reactive derivative thereof.

Expression

Another convenient way of producing a peptidyl molecule according to the present invention (peptide or polypeptide) is to express nucleic acid encoding it, by use of nucleic acid in an expression system. Accordingly the present invention also provides in various aspects nucleic acid encoding the polypeptides and peptides of the invention.

Generally, nucleic acid according to the present invention is provided as an isolate, in isolated and/or purified form, or free or substantially free of material with which it is naturally associated, such as free or substantially free of nucleic acid flanking the gene in the human genome, except possibly one or more regulatory sequence(s) for expression.

Nucleic acid may be wholly or partially synthetic and may include genomic DNA, cDNA or RNA. Where nucleic acid according to the invention includes RNA, reference to the sequence shown should be construed as reference to the RNA equivalent, with U substituted for T.

Nucleic acid sequences encoding a polypeptide or peptide in accordance with the present invention can be readily prepared by the skilled person using the information and references contained herein and techniques known in the art (for example, see Sambrook, Fritsch and Maniatis, "Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1989, and Ausubel et al, Short Protocols in Molecular Biology, John Wiley and Sons, 1992), given the nucleic acid sequence and clones available. These techniques include (i) the use of the polymerase chain reaction (PCR) to amplify samples of such nucleic acid, e.g. from genomic sources, (ii) chemical synthesis, or (iii) preparing cDNA sequences. DNA encoding p21 fragments may be generated and used in any suitable way known to those of skill in the art, including by taking encoding DNA, identifying suitable restriction enzyme recognition sites either side of the portion to be expressed, and cutting out said portion from the DNA. The portion may then be operably linked to a suitable promoter in a standard commercially available expression system. Another recombinant approach is to amplify the relevant portion of the DNA with suitable PCR primers. Modifications to the Cdc25A sequences can be made, e.g. using site directed mutagenesis, to lead to the expression of modified Cdc25A peptide or to take account of codon preference in the host cells used to express the nucleic acid.

In order to obtain expression of the nucleic acid sequences, the sequences can be incorporated in a vector having one or more control sequences operably linked to the nucleic acid to control its expression. The vectors may include other sequences such as promoters or enhancers to drive the expression of the inserted nucleic acid, nucleic acid sequences so that the polypeptide or peptide is produced as a fusion and/or nucleic acid encoding secretion signals so that the polypeptide produced in the host cell is secreted from the cell. Polypeptide can then be obtained by transforming the vectors into host cells in which the vector is functional, culturing the host cells so that the polypeptide is produced and recovering the polypeptide from the host cells or the surrounding medium. Prokaryotic and eukaryotic cells are used for this purpose in the art, including strains of E. coli, yeast, and eukaryotic cells such as COS or CHO cells.

Accordingly, the present invention also encompasses a method of making a polypeptide or peptide, the method including expression from nucleic acid encoding the polypeptide or peptide. This may conveniently be achieved by growing a host cell in culture, containing such a vector, under appropriate conditions which cause or allow expression of the polypeptide. Polypeptides and peptides may also be expressed in in vitro systems, such as reticulocyte lysate.

Suitable vectors can be chosen or constructed, containing appropriate regulatory sequences, including promoter sequences, terminator fragments, polyadenylation sequences, enhancer sequences, marker genes and other sequences as appropriate. Vectors may be plasmids, viral e.g. 'phage, or phagemid, as appropriate. For further details see, for example, Molecular Cloning: a Laboratory Manual: 2nd edition, Sambrook et al., 1989, Cold Spring Harbor Laboratory Press. Many known techniques and protocols for manipulation of nucleic acid, for example in preparation of nucleic acid constructs, mutagenesis, sequencing, introduction of DNA into cells and gene expression, and analysis of proteins, are described in detail in Current Protocols in Molecular Biology, Ausubel et al. eds., John Wiley & Sons, 1992.

Systems for cloning and expression of a polypeptide in a variety of different host cells are well known. Suitable host cells include bacteria, eukaryotic cells such as mammalian and yeast, and baculovirus systems. Mammalian cell lines available in the art for expression of a heterologous polypeptide include Chinese hamster ovary cells, HeLa cells, baby hamster kidney cells, COS cells, U-2-OS cells, SAOS-2 cells and many others. A common, preferred bacterial host is E. coli.

Thus, a further aspect of the present invention provides a host cell containing heterologous nucleic acid as disclosed herein.

The nucleic acid of the invention may be integrated into the genome (e.g. chromosome) of the host cell. Integration may be promoted by inclusion of sequences which promote recombination with the genome, in accordance with standard techniques. The nucleic acid may be on an extra-chromosomal vector within the cell, or otherwise identifϊably heterologous or foreign to the cell.

A still further aspect provides a method which includes introducing the nucleic acid into a host cell. The introduction, which may (particularly for in vitro introduction) be generally referred to without limitation as "transformation", may employ any available technique. For eukaryotic cells, suitable techniques may include calcium phosphate transfection, DEAE-Dextran, electroporation, liposome-mediated transfection and transduction using retrovirus or other virus, e.g. vaccinia or, for insect cells, baculovirus. For bacterial cells, suitable techniques may include calcium chloride transformation, electroporation and transfection using bacteriophage. As an alternative, direct injection of the nucleic acid could be employed.

Marker genes such as antibiotic resistance or sensitivity genes may be used in identifying clones containing nucleic acid of interest, as is well known in the art.

The introduction may be followed by causing or allowing expression from the nucleic acid, e.g. by culturing host cells (which may include cells actually transformed although more likely the cells will be descendants of the transformed cells) under conditions for expression of the gene, so that the encoded polypeptide (or peptide) is produced. If the polypeptide is expressed coupled to an appropriate signal leader peptide it may be secreted from the cell into the culture medium. Following production by expression, a polypeptide or peptide may be isolated and/or purified from the host cell and/or culture medium, as the case may be, and subsequently used as desired, e.g. in the formulation of a composition which may include one or more additional components, such as a pharmaceutical composition which includes one or more pharmaceutically acceptable excipients, vehicles or carriers.

Introduction of nucleic acid encoding a peptidyl molecule according to the present invention may take place in vivo by way of gene therapy, to enhance or promote the interaction between Cdc25A, cyclin B, Cdkl, or cyclin B/Cdkl, or to disrupt or interfere with the interaction of cyclinB, Cdkl, or cyclin B/Cdkl with Weel or Mytl. .

Thus, a host cell containing nucleic acid according to the present invention, e.g. as a result of introduction of the nucleic acid into the cell or into an ancestor of the cell and/or genetic alteration of the sequence endogenous to the cell or ancestor (which introduction or alteration may take place in vivo or ex vivo), may be comprised (e.g. in the soma) within an organism which is an animal, particularly a mammal, which may be human or non-human, such as rabbit, guinea pig, rat, mouse or other rodent, cat, dog, pig, sheep, goat, cattle or horse, or which is a bird, such as a chicken. Genetically modified or transgenic animals or birds comprising such a cell are also provided as further aspects of the present invention.

This procedure may have a therapeutic aim. Also, the presence of a mutant, allele, derivative or variant sequence within cells of an organism, particularly when in place of a homologous endogenous sequence, may allow the organism to be used as a model in testing and/or studying compositions which modulate activity of the encoded polypeptide in vitro or are otherwise indicated to be of therapeutic potential. Conveniently, however, assays for such compositions may be carried out in vitro, within host cells or in cell-free systems.

Suitable screening methods are conventional in the art. They include techniques such as radioimmunosassay, scintillation proximetry assay and ELISA methods. Suitably either the Cdc25A protein or cyclin B or Cdkl or cyclin B/Cdkl, or a fragment, an analogue, derivative, variant or functional mimetic of any of these protein, is immobilized whereupon the other is applied in the presence of the agents under test. In a scintillation proximetry assay a biotinylated protein fragment is bound to streptavidin coated scintillant - impregnated beads (produced by Amersham). Binding of radiolabeled peptide is then measured by determination of radioactivity induced scintillation as the radioactive peptide binds to the immobilized fragment. Agents which intercept this are thus inhibitors of the interaction.

Alternatively, the phosphorylation of Cdc25A, particularly on Serl7 or Serl 15, or the dephosphorylation of Cdkl, particularly on Thrl4 or Tyrl5, may be measured, such as by incorporation or removal of labeled phosphates, asobserved by a signal. Signalling may be observed in a variety of ways known in the art, including radioisotopic, chemical, fluorescent, and enzymatic signaling. Alternatively, the number of mitotic cells in a sample may be measured, such as by flow cytometry, microscopic techniques, visualization, or other techniques known in the art. For example, flow cytometry measurements may involve staining of chromosomes with phospho-histone (H3), a marker of productive entry into mitosis. Alternatively, a light or electron microscope may be used to measure G2/M phase transition. Screening may be high-throughput or low-throughput.

Assays

In one general aspect, the present invention provides ause of the interaction of Cdc25A and cyclin B/Cdkl for screening for compositions which are capable of modulating the interaction of Cdc25A and cyclin B/Cdkl . This may involve using the compositions described above identifying binding partners of the composition. Alternatively, it may involve compounds having the property of binding to, or enhancing the activity of, cyclin B/Cdkl and promoting the phosphorylation of Cdc25A. For example, a composition may activate cyclin B/Cdkl by dephosphorylating Cdkl on Thr 14 or Tyrl5, or by inhibiting phosphorylation of Thrl4 (such as by inhibiting Mytl) or Tyr 15 (such as by inhibiting the Weel kinase). Similarly, it may involve compositions that phosphorylate, or trigger the phosphorylation of, Cdc25A (stable form), particularly at Serl7 or Serl 15.

In further embodiments of the invention, these methods may employ sequence variants of the above mentioned Cdc25A sequences, or fragments thereof, e.g. peptides in which Serl7 or Serl 15 is substituted by a residue such as alanine which is non-phosphorlyatable or resists phosphorylation, or by a residue such glutamic acid which mimics phosphorylation.

Phosphorylated peptides for raising antibodies

The peptide fragments of the invention may be linked by any convenient covalent bond, preferably to the N-terminus, to a coupling partner consisting preferably of a small peptide sequence to form a peptide conjugate. The term "peptide conjugate" as used herein indicates a fusion between at least two peptide sequences via a peptidic bond or an equivalent bioisosteric bond, such as the peptide bond mimetics described in Table 1 in Tayar et al., Amino Acids (1995) 8:125-139. Said coupling partner is preferably selected from the group consisting of an HIV tat peptide residues 49-57, HIV tat peptide residues 49-56, the tat sequence YGRKK-RRQRRR, a polyarginine peptide having from 6 to 20 residues, such as Rg , and transducing peptide sequences which are able to maintain sufficient levels of peptide conjugate within cells and which does not interfere with the folding of the peptide fragment, such as the following peptide sequences: YARKARRQARR, YARAAARQARA, YARAARRAARR, YARAARRAARA, ARRRRRRRRR, and YAAARRRRRRR, which are disclosed in WO 99/29721 and in US patent No. 6,221,355 (seq. id. nos. 3-8) the disclosures of which are incorporated herein by reference.

The peptide fragments used herein or useful in the methods described herein consist preferably of a sequence of about 11 amino acid residues corresponding to a sequence of Cdc25A around one of the Serl7 or Serl 15 residues positioned in the middle of the sequence. The serine residue may be substituted with a serine analogue, i.e. an amino acid having properties similar to serine, or, preferably resembling serine but being unphosphorylatable. Examples of serine analogues are alanine, L-Abu (L-2-aminobutanoic acid), Aib (2- aminoisobutanoic acid), beta-alanine, Aoa (aminooxyacetic acid), Val, Leu, L-Nva (L-2- aminovaleric acid), Sar (sarcosine) and He. An advantage of substituting serine with an unnatural amino acid residue is a possible protection of the peptide fragment against proteolytic degradation. A idation of the C-terminus also reduces susceptibility to proteolytic degradation and is preferred in the peptide fragments of the invention.

In both of these methods, modulation of the Cdc25A with cyclin B/Cdkl interaction by a candidate compound can be assessed by determining the presence or extent of the binding or disruption of Cdc25A to cyclin B/Cdkl, by determining the presence or extent of the phosphorylation of Cdc25A by cyclin B/Cdkl, or by determining the presence or amount of Cdc25A present in a cell based assay. Alternatively, a candidate compound can also be assessed by determining the presence or extent of non-phosphorylation or dephosphorylation of Cdkl . These determinations can be combined with the determination of whether the candidate compound is capable of promoting G2/M transition (entry into mitosis) in a population of cells, e.g. in cell based assay.

Performance of an assay method according to the present invention may be followed by isolation and/or manufacture and/or use of a compound, composition or molecule which tests positive for ability to promote or enhance interaction between Cdc25A and cyclin B/Cdkl and/or promote the phosphorylation of Cdc25A bycyclin B/Cdkl.

In carrying out these methods, it may be convenient to screen a plurality of candidate compounds, e.g. as present in a library, at the same time, e.g. by contacting a mixture of different candidate compounds with the interacting peptides, and then in the event of a positive result resolving which member of the mixture is active. These technique are used in high throughput screening (HTS) to increase the numbers of compounds, e.g. resulting from combinatorial chemistry program or present in library derived from a natural source material, which can be screened in a method. The precise format of the assays of the invention may be varied by those of skill in the art using routine skill and knowledge. For example, interaction between compositions may be studied in vitro by labeling one with a detectable label and bringing it into contact with the other which has been immobilised on a solid support. Suitable detectable labels, especially for peptidyl substances include ³⁵S-methionine which may be incorporated into recombinantly produced peptides and polypeptides. Recombinantly produced peptides and polypeptides may also be expressed as a fusion protein containing an epitope which can be labeled with an antibody. Fusions can also be used to display the peptide fragments of Cdc25A, cyclin B, or Cdkl as an inserted motif, e.g. in a protein such as thioredoxin, in order to present the peptide motifs in a correct three dimensional structure.

The protein which is immobilized on a solid support may be immobilized using an antibody against that protein bound to a solid support or via other technologies which are known per se. A preferred in vitro interaction may utilize a fusion protein including glutathione-S-transferase (GST). This may be immobilized on glutathione agarose beads. In an in vitro assay format of the type described above a test compound can be assayed by determining its ability to diminish the amount of labeled peptide or polypeptide which binds to the immobilized GST-fusion polypeptide. This may be determined by fractionating the glutathione-agarose beads by SDS-polyacrylamide gel electrophoresis. Alternatively, the beads may be rinsed to remove unbound protein and the amount of protein which has bound can be determined by counting the amount of label present in, for example, a suitable scintillation counter.

In one embodiment, the screening method looks for small molecules in the chemical compound libraries, which would promote phosphorylation of the Cdc25A fragment, e.g. a GST-fusion protein with a fragment of Cdc25A or an analogous peptide. The assay detects the extent to which the candidate compounds modulate (e.g. promote) the phosphorlyation of the amino acid corresponding to Serl7 and/or Serl 15 in full length Cdc25A. In one preferred format, the assay employs a solid phase such as a plastic well plate, on which the GST- Cdc25A fragment can be immobilised at one or more locations. The Cdc25A can then be exposed to cyclin B/Cdkl, which would normally phosphorylate the Cdc25A peptide. This can be readily detected either by incorporation of radioactive phosphate into the peptide and counting radioactivity, or by using a phosphospecific antibody against Serl7 or Serl 15, produced as using one of the peptides of the invention as discussed above. The detection step can be carried out after washing off the Chk from the plate and the use of the antibody has the advantage of being very specific and non-radioactive.

Alternatively, the inhibitory compounds identified using the assay of the invention might be expected to fall into at least two possible categories: direct inhibitors of cyclin B/Cdkl, or those which interfere with the recognition and/or phosphorylation of cyclin B/Cdkl . Both types of compounds might be useful for the present invention and might be subject to further characterization.

An assay according to the present invention may also take the form of an in vivo assay. The in vivo assay may be performed in a cell line such as a yeast strain or mammalian cell line in which the relevant polypeptides or peptides are expressed from one or more vectors introduced into the cell.

The amount of candidate composition or compound which may be added to an assay of the invention will normally be determined by trial and error depending upon the type of compound used. Typically, from about 0.01 to 100 nM concentrations of putative inhibitor compound may be used, for example from 0.1 to 10 nM. Greater concentrations may be used when a peptide is the test substance/composition/compound.

Compounds which may be used may be natural or synthetic chemical compounds used in drug screening programs. Extracts of plants which contain several characterized or uncharacterized components may also be used.

Antibodies

Antibodies directed to the site of interaction in either protein form a further class of putative inhibitor compounds. As mentioned above, the present invention further provides the use of the peptides and compositions of the present invention for raising antibodies and in particular for raising antibodies which are capable of recognizing Cdc25A phosphorylation sites in either a phosphorylated or dephosphorylated form. Likewise, the present invention further provides the use of the peptides and compositions of the present invention for raising antibodies and in particular for raising antibodies which are capable of recognizing Cdkl phosphorylation sites in either a phosphorylated or dephosphorylated form. Candidate inhibitor antibodies may be characterized and their binding regions determined to provide single chain antibodies and fragments thereof which are responsible for disrupting the interaction. Methods for producing various types of antibodies are well-known in the art (e.g., Harlow & Lane (1988) Antibodies: A General Laboratory Method Cold Spring Harbor).

Antibodies may be obtained using techniques which are standard in the art. Methods of producing antibodies include immunizing a mammal with the protein or a fragment thereof. Antibodies may be obtained from immunized animals using any of a variety of techniques known in the art, and screened, preferably using binding of antibody to antigen of interest. For instance, Western blotting techniques or immunoprecipitation may be used (Armitage et al, Nature 357:80-82, 1992). Isolation of antibodies and/or antibody-producing cells from an animal may be accompanied by a step of sacrificing the animal.

As an alternative or supplement to immunizing a mammal with a peptide, an antibody specific for a protein may be obtained from a recombinantly produced library of expressed immunoglobulin variable domains, e.g. using lambda bacteriophage or filamentous bacteriophage which display functional immunoglobulin binding domains on their surfaces; for instance see WO92/01047. The library may be naive, that is constructed from sequences obtained from an organism which has not been immunized with any of the proteins (or fragments), or may be one constructed using sequences obtained from an organism which has been exposed to the antigen of interest.

Antibodies according to the present invention may be modified in a number of ways. Indeed the term "antibody" should be construed as covering any binding substance having a binding domain with the required specificity. Thus, the invention covers antibody fragments, derivatives, functional equivalents and homologues of antibodies, including synthetic molecules and molecules whose shape mimics that of an antibody enabling it to bind an antigen or epitope.

A hybridoma producing a monoclonal antibody according to the present invention may be subject to genetic mutation or other changes. It will further be understood by those skilled in the art that a monoclonal antibody can be subjected to the techniques of recombinant DNA technology to produce other antibodies or chimeric molecules which retain the specificity of the original antibody. Such techniques may involve introducing DNA encoding the immunoglobulin variable region, or the complementarity determining regions (CDRs), of an antibody to the constant regions, or constant regions plus framework regions, of a different immunoglobulin. See, for instance, EP 0 184 187 A, GB 2 188 638 A or EP 0 239 400 A. Cloning and expression of chimeric antibodies are described in EP 0 120 694 A and EP 0 125 023 A.

Hybridomas capable of producing antibody with desired binding characteristics are within the scope of the present invention, as are host cells, eukaryotic or prokaryotic, containing nucleic acid encoding antibodies (including antibody fragments) and capable of their expression. The invention also provides methods of production of the antibodies including growing a cell capable of producing the antibody under conditions in which the antibody is produced, and preferably secreted.

The reactivities of antibodies on a sample may be determined by any appropriate means. Tagging with individual reporter molecules is one possibility. The reporter molecules may directly or indirectly generate detectable, and preferably measurable, signals. The linkage of reporter molecules may be directly or indirectly, covalently, e.g. via a peptide bond or non- covalently. Linkage via a peptide bond may be as a result of recombinant expression of a gene fusion encoding antibody and reporter molecule.

One favored mode is by covalent linkage of each antibody with an individual fluorochrome, phosphor or laser dye with spectrally isolated absorption or emission characteristics. Suitable fluorochromes include fluorescein, rhodamine, phycoerythrin and Texas Red. Suitable chromogenic dyes include diaminobenzidine.

Other reporters include macromolecular colloidal particles or particulate material such as latex beads that are colored, magnetic or paramagnetic, and biologically or chemically active agents that can directly or indirectly cause detectable signals to be visually observed, electronically detected or otherwise recorded. These molecules may be enzymes which catalyze reactions that develop or change colors or cause changes in electrical properties, for example. They may be molecularly excitable, such that electronic transitions between energy states result in characteristic spectral absorptions or emissions. They may include chemical entities used in conjunction with biosensors. Biotin/avidin or biotin/streptavidin and alkaline phosphatase detection systems may be employed.

The mode of determining binding is not a feature of the present invention and those skilled in the art are able to choose a suitable mode according to their preference and general knowledge.

Antibodies may also be used in purifying and/or isolating a polypeptide or peptide according to the present invention, for instance following production of the polypeptide or peptide by expression from encoding nucleic acid therefore. Antibodies may be useful in a therapeutic context (which may include prophylaxis) to disrupt cyclin B/Cdkl interaction with Weel, Mytl, or another enzyme with a view to inhibiting Cdkl and/or cyclin B phosphorylation. Antibodies can for instance be microinjected into cells.

Mimetic Compounds

Other candidate inhibitor compounds may be based on modeling the 3 -dimensional structure of a polypeptide or peptide fragment and using rational drug design to provide potential inhibitor compounds with particular molecular shape, size and charge characteristics.

Following identification of a substance or agent which modulates or affects the phosphorylation of cyclin B/Cdkl by Weel, Mytl or another enzyme, the substance or agent may be investigated further.

As noted, the agent may be peptidyl, e.g., a peptide which includes a sequence as recited above, or may be a functional analogue of such a peptide.

As used herein, the expression "functional analogue" relates to peptide variants or organic compounds having the same functional activity as the peptide in question.

Suitable modeling techniques are known in the art. This includes the design of so- called "mimetics" which involves the study of the functional interactions fluorogenic oligonucleotide the molecules and the design of compounds which contain functional groups arranged in such a manner that they could reproduced those interactions.

The designing of mimetics to a known pharmaceutically active compound is a known approach to the development of pharmaceuticals based on a lead compound. This might be desirable where the active compound is difficult or expensive to synthesize or where it is unsuitable for a particular method of administration, e.g. peptides are not well suited as active agents for oral compositions as they tend to be quickly degraded by proteases in the alimentary canal. Mimetic design, synthesis and testing may be used to avoid randomly screening large number of molecules for a target property.

There are several steps commonly taken in the design of a mimetic from a compound having a given target property. Firstly, the particular parts of the compound that are critical and/or important in determining the target property are determined. In the case of a peptide, this can be done by systematically varying the amino acid residues in the peptide, e.g. by substituting each residue in turn. These parts or residues constituting the active region of the compound are known as its "pharmacophore".

Once the pharmacophore has been found, its structure is modeled to according its physical properties, e.g. stereochemistry, bonding, size and/or charge, using data from a range of sources, e.g. spectroscopic techniques, X-ray diffraction data and NMR. Computational analysis, similarity mapping (which models the charge and/or volume of a pharmacophore, rather than the bonding between atoms) and other techniques can be used in this modeling process.

In a variant of this approach, the three-dimensional structure of the ligand and its binding partner are modeled. This can be especially useful where the ligand and/or binding partner change conformation on binding, allowing the model to take account of this in the design of the mimetic.

A template molecule is then selected onto which chemical groups which mimic the pharmacophore can be grafted. The template molecule and the chemical groups grafted on to it can conveniently be selected so that the mimetic is easy to synthesize, is likely to be pharmacologically acceptable, and does not degrade in vivo, while retaining the biological activity of the lead compound. The mimetic or mimetics found by this approach can then be screened to see whether they have the target property, or to what extent they exhibit it. Further optimization or modification can then be carried out to arrive at one or more final mimetics for further testing or optimization, e.g. in vivo or clinical testing. The mimetic or mimetics found by this approach can then be screened to see whether they have the target property, or to what extent they exhibit it. Further optimization or modification can then be carried out to arrive at one or more final mimetics for in vivo or clinical testing.

Mimetics of this type together with their use in therapy form a further aspect of the invention.

The present invention further provides the use of a peptide which includes a sequence as disclosed, or a derivative, active portion, analogue, variant or mimetic, thereof able to bindcyclin B/Cdkl, in screening for a composition able to bind Cdc25A and/or having the activity of promoting the phosphorylation of Cdc25A, e.g. at Serl7 or Serl 15.

Pharmaceutical Uses

The compositions of the invention can be used in the treatment cancer and other hyperproliferative disorders such as psoriasis, arteriogenesis or inflammation, and in particular in the treatment of conditions in which the accumulation of Cdc25A at the G2/M transition can be employed to sensitized diseased cells to a further treatment such as chemotherapy or radiotherapy. Substances or compositions described in the application can be used individually or in various combinations.

In general, the aim of the combination of the chemotherapy or radiotherapy and the compositions and uses of the present invention is to reduce the amount or frequency of the therapies, many of which have serious side effects for a patient, or to enhance the effectiveness of a given therapy, e.g. in the proportion of diseased cells in a population which are killed or commit to apoptosis as a result of the treatment.

Examples of chemotherapeutic agents which can be employed in combination with the compositions of the invention include DNA topoisomerase inhibitors, e.g. DNA topoisomerase I toxins such as Camptothecin and derivatives such as topotecan; DNA topoisomerase II toxins such as anthracyclines such as Daunorubicin, Doxorubicin and Adriamycin and epipodophyllotoxines such as etoposide and teniposide. Examples of radiotherapy include treatment with γ-radiation and X-rays.

Generally, a composition according to the present invention is provided in an isolated and/or purified form. This may include being in a further composition where it represents at least about 90% active ingredient, more preferably at least about 95%, more preferably at least about 98%. Such a composition may, however, include inert carrier materials or other pharmaceutically and physiologically acceptable excipients. As noted below, a composition according to the present invention can include in addition to an inhibitor compound as disclosed, one or more other molecules of therapeutic use, such as an anti-tumor agent.

The present invention extends in various aspects not only to a substance identified as a modulator of Cdc25A and cyclin B/Cdkl interaction or activity, property or pathway in accordance with what is disclosed herein, but also a pharmaceutical composition, medicament, drug or other composition comprising such a substance, a method comprising administration of such a composition to a patient, e.g. for anti-cancer, use of such a substance in manufacture of a composition for administration, e.g. for the treatment of cancer, and a method of making a pharmaceutical composition comprising admixing such a substance/composition with a pharmaceutically acceptable excipient, vehicle or carrier, and optionally other ingredients.

A substance/composition according to the present invention such as a promoter of Cdc25A and cyclin B/Cdkl interaction or binding may be provided for use in a method of treatment.

The invention further provides a method of enhancing or otherwise modulating cyclin B/Cdkl activity, or other Cdc25A-mediated activity in a cell, which includes administering an agent which enhances the binding of Cdc25A to cyclin B/Cdkl protein, such a method being useful in treatment of cancer and other hyperproliferative disorders.

The hyperproliferative disorder being treated or prevented is cancer, arteriogenesis, psoriasis, angiogenesis, inflammation, or nonmalignant tumor. Other hyperproliferative disorders (e.g., skin tags and other dermatological disorders) readily suggest themselves to one of ordinary skill in the art.

Whether it is a polypeptide, antibody, peptide, nucleic acid molecule, small molecule, mimetic or other pharmaceutically useful compound according to the present invention that is to be given to an individual, administration is preferably in a "prophylactically effective amount" or a "therapeutically effective amount" (as the case may be, although prophylaxis may be considered therapy), this being sufficient to show benefit to the individual. The actual amount administered, and rate and time-course of administration, will depend on the nature and severity of what is being treated. Prescription of treatment, e.g. decisions on dosage etc, is within the responsibility of general practitioners and other medical doctors.

Pharmaceutical compositions according to the present invention, and for use in accordance with the present invention, may include, in addition to active ingredient, a pharmaceutically acceptable excipient, carrier, buffer, stabilizer or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient. The precise nature of the carrier or other material will depend on the route of administration, which may be oral, or by injection, e.g. cutaneous, subcutaneous or intravenous.

Pharmaceutical compositions for oral administration may be in tablet, capsule, powder or liquid form. A tablet may include a solid carrier such as gelatin or an adjuvant. Liquid pharmaceutical compositions generally include a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oil. Physiological saline solution, dextrose or other saccharide solution or glycols such as ethylene glycol, propylene glycol or polyethylene glycol may be included.

For intravenous, cutaneous or subcutaneous injection, or injection at the site of affliction, the active ingredient will be in the form of a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability. Those of relevant skill in the art are well able to prepare suitable solutions using, for example, isotonic vehicles such as sodium chloride injection, Ringer's injection, lactated Ringer's injection. Preservatives, stabilizers, buffers, antioxidants and/or other additives may be included, as required.

Examples of techniques and protocols mentioned above can be found in Remington's Pharmaceutical Sciences, 16th edition, Osol, A. (ed), 1980. The agent may be administered in a localized manner to a tumor site or other desired site or may be delivered in a manner in which it targets tumor or other cells.

Targeting therapies may be used to deliver the active agent more specifically to certain types of cell, by the use of targeting systems such as antibody or cell specific ligands. Targeting may be desirable for a variety of reasons, for example if the agent is unacceptably toxic, or if it would otherwise require too high a dosage, or if it would not otherwise be able to enter the target cells.

Instead of administering these agents directly, they may be produced in the target cells by expression from an encoding gene introduced into the cells, e.g. in a viral vector (a variant of the VDEPT technique - see below). The vector may targeted to the specific cells to be treated, or it may contain regulatory elements which are switched on more or less selectively by the target cells.

The agent maybe administered in a precursor form, for conversion to the active form by an activating agent produced in, or targeted to, the cells to be treated. This type of approach is sometimes known as ADEPT or VDEPT, the former involving targeting the activating agent to the cells by conjugation to a cell-specific antibody, while the latter involves producing the activating agent, e.g. an enzyme, in a vector by expression from encoding DNA in a viral vector (see for example, EP 0 415 731 A and WO90/07936).

A composition may be administered alone or in combination with other treatments, either simultaneously or sequentially dependent upon the condition to be treated, such as cancer, virus infection or any other condition in which a Cdc25A mediated effect is desirable.

Nucleic acid according to the present invention, encoding a polypeptide or peptide able to enhance Cdc25A and cyclin B/Cdkl interaction or binding, Cdc25A phosphorylation, cyclin B/Cdkl dephosphorylation, or other Cdc25A-mediated cellular pathway or function, may be used in methods of gene therapy, for instance in treatment of individuals with the aim of preventing or curing (wholly or partially) cancer.

Vectors such as viral vectors have been used in the prior art to introduce nucleic acid into a wide variety of different target cells. Typically the vectors are exposed to the target cells so that transfection can take place in a sufficient proportion of the cells to provide a useful therapeutic or prophylactic effect from the expression of the desired polypeptide. The transfected nucleic acid may be permanently incorporated into the genome of each of the targeted tumor cells, providing long lasting effect, or alternatively the treatment may have to be repeated periodically.

A variety of vectors, both viral vectors and plasmid vectors, are known in the art, see US Patent No. 5,252,479 and WO93/07282. In particular, a number of viruses have been used as gene transfer vectors, including papovaviruses, such as SV40, vaccinia virus, herpesviruses, including HSV and EBV, and retroviruses. Many gene therapy protocols in the prior art have used disabled murine retroviruses.

As an alternative to the use of viral vectors other known methods of introducing nucleic acid into cells includes electroporation, calcium phosphate co-precipitation, mechanical techniques such as microinjection, transfer mediated by liposomes and direct DNA uptake and receptor-mediated DNA transfer.

Receptor-mediated gene transfer, in which the nucleic acid is linked to a protein ligand via polylysine, with the ligand being specific for a receptor present on the surface of the target cells, is an example of a technique for specifically targeting nucleic acid to particular cells.

A polypeptide, peptide or other substance able to interfere with the interaction of the relevant polypeptide, peptide or other substance as disclosed herein, or a nucleic acid molecule encoding a peptidyl such molecule, may be provided in a kit, e.g. sealed in a suitable container which protects its contents from the external environment. Such a kit may include instructions for use.

As described above, the invention provides a method for treating or preventing cancer or a hyperproliferative disorder comprising administering a composition which is capable of enhancing the interaction Of Cdc25A and cyclinB/Cdkl . In particular, the composition enhances the binding of Cdc25A to cyclin B/Cdkl or the phosphorylation of Cdc25A by cyclin B/Cdkl. Examples of the hyperproliferative disorder include psoriasis, arteriogenesis, angiogenesis, inflammation, or non-malignant tumor. It is within the scope of the invention that substances or compositions described in the application can be used individually or in various combinations.

In another embodiment, the composition is administered in combination with chemotherapy or radiotherapy. Preferably, the chemotherapy includes a proteasome inhibitor, such as LLnL, or a functional derivative thereof, or the chemotherapy includes caffeine or a functional derivative thereof. Alternatively, the chemotherapy includes cyclin B/Cdkl kinase or a functional derivative thereof; Cdc25A that is phosphorylated on at least one of Serl 7 or Serl 15, or a functional derivative thereof; or the administration of a DNA topoisomerase toxin, such as an etoposide, an anthracycline, an epipodophyllotoxine or a camptothecin derivative.

In a preferred embodiment, the radiotherapy includes the use of γ-radiation.

hi a one embodiment, the composition comprises a protein complex comprising:

(a) a first protein moiety comprising:

(i) a protein which has at least 80% sequence identity with a corresponding sequence of cyclin B; or

(ii) a derivative of protein (ai); or

(iii) a substance which is protein (ai) or derivative (aii) linked to a coupling partner; and

(b) a second protein moiety comprising:

(i) a protein which has at least 80% sequence identity with a corresponding sequence of Cdkl; or

(ii) a derivative of protein (bi); or

(iii) a substance which is protein (bi) or derivative (bii) linked to a coupling partner.

In another embodiment, the composition comprises:

(a) a protein which has at least 80% sequence identity with a corresponding sequence of Cdc25A, the protein including a serine residue at a position corresponding to amino acid Serl 7 or Ser 115 in Cdc25A; or,

(b) a derivative of protein (a); or

(c) a substance which is protein (a) or derivative (b) linked to a coupling partner. i another embodiment, the composition comprises cyclin B/Cdkl or a functional derivative thereof capable of phosphorylating Cdc25A at Serl7 or Serl 15.

In another embodiment, the composition comprises Cdc25A or a functional derivative thereof, capable of enhancing the activity of cyclin B/Cdkl kinase.

In yet another embodiment, the method comprises

(a) the composition comprises Cdc25A or a functional derivative thereof, including a phosphorylated serine residue at a position corresponding to amino acid Serl7 or Ser 115 in Cdc25A; and

(b) the composition is capable of enhancing the activity of cyclin B/Ckdl kinase.

Preferably, (a) the cyclin B comprises a mammalian cyclin B; or (b) the Cdkl comprises a mammalian Cdkl. Preferably, the Cdc25A comprises as mammalian Cdc25A.

In a preferred embodiment, the composition comprises an inhibitor of proteasome degradation. Preferably, the inhibitor comprises LLnL or a functional derivative thereof. Preferably, the composition comprises caffeine or a functional derivative thereof.

In a preferred embodiment, the method further comprises administering the composition to a mammal in an amount sufficient to increase the level of Cdc25 A by at least 10% as determined by a standard protein assay.

In another aspect, the invention provides a composition having the property of promoting the phosphorylation of Cdc25A or a derivative thereof, wherein the Cdc25A or the derivative thereof, includes a serine residue at a position corresponding to amino acid Serl7 or Serl 15 in Cdc25A, and wherein the composition comprises a protein complex comprising: (a) a first protein moiety comprising:

(i) a protein which has at least 80% sequence identity with a corresponding sequence of cyclin B; or

(ii) a derivative of protein (ai); or

(iii) a substance which is protein (ai) or derivative (aii) linked to a coupling partner; and (b) a second protein moiety comprising:

(i) a protein which has at least 80% sequence identity with a corresponding sequence of Cdkl; or

(ii) a derivative of protein (bi); or

(iii) a substance which is protein (bi) or derivative (bii) linked to a coupling partner.

In another aspect, the invention provides a composition having the property of enhancing the activity of cyclin B/Cdkl and promoting the phosphorylation of Cdc25 A by the cyclin B/Cdkl, the composition comprising:

(a) a protein which has at least 80% sequence identity with a corresponding sequence of Cdc25A, the protein including a serine residue at a position corresponding to amino acid Serl7 or Serl 15 in Cdc25A; or,

(b) a derivative of protein (a); or

(c) a substance which is protein (a) or derivative (b) linked to a coupling partner.

The invention also provides an isolated nucleic acid molecule encoding the composition; an expression vector comprising the nucleic acid, operably linked to sequences to direct its expression; a host cell transformed with the expression vector; a method of producing the composition above, the method comprising culturing the host cells above and isolating the composition thus produced; use of the composition above in a method of medical treatment; and a pharmaceutical composition comprising the composition above.

The invention also provides a method for using the composition above for identifying (i) binding partners of the composition or (ii) compounds having the property of enhancing the activity of cyclin B/Cdkl kinase and promoting the phosphorylation of Cdc25A.

In another aspect, the invention provides a method of identifying compounds capable of modulating the interaction of Cdc25A and cyclin B/Cdkl kinase, the method comprising:

(a) contacting (i) a composition comprising Cdc25A or a fragment or variant thereof, (ii) a composition comprising cyclin B/Cdkl kinase or a fragment or variant thereof and (iii) a candidate compound, under conditions wherein, in the absence of the candidate compound, the compositions interact; and

(b) determining the interaction between the compositions to identify whether the candidate compound modulates the interaction.

In one embodiment, the interaction determined in step (b) is the binding of Cdc25A by cyclin B/Cdkl kinase. Alternatively, the interaction determined in step (b) is the phosphorylation of Cdc25A by cyclin B/Cdkl kinase, or the presence or amount of Cdc25A present in a cell based assay.

In one embodiment, the method above uses a compound capable of modulating the interaction of Cdc25A and cyclin B/Cdkl kinase is capable of enhancing the interaction or the phosphorylation of Cdc25A by cyclin B/Cdkl kinase. In one example, the Cdc25A is fusion of GST and a fragment of Cdc25A comprising an amino acid sequence corresponding to the Ser 17 or Serl 15 of full length Cdc25A.

In one embodiment, the method comprises determining the modulation of the interaction of Cdc25A and cyclin B/Cdkl by measuring the phosphorylation of the Cdc25A peptide, such as by the incorporation of radioactive phosphate into the Cdc25A peptide, or by using an antibody capable of specifically binding to phosphorylated Cdc25 A peptide.

In addition, the method may further comprise testing a candidate compound identified in step (b) to determine whether it is capable of promoting transition from G2 phase into M phase in a population of cells.

In another aspect, the invention provides a method of identifying binding partners of a composition having the property of enhancing the activity of cyclin B/Cdkl kinase and promoting the phosphorylation of Cdc25 A by the cyclin B/Cdkl kinase, the composition comprising a protein having at least 80% sequence identity with a corresponding sequence of Cdc25A, the protein including serine at a position corresponding to amino acid Serl 7 or Serl 15 in Cdc25A, the method comprising contacting the composition and a candidate compound and determining whether the candidate compound has the property of binding to the composition.

Preferably, the method further comprises testing the compounds which bind to Cdc25A for activity in promoting the phosphorylation of Cdc25A by cyclin B/Cdkl kinase, or testing the candidate compound to determine whether it is capable of promoting transition from G2 phase into M phase in a population of cells.

In a preferred embodiment, a plurality of candidate compounds are contacted with the compositions. Preferably, the plurality of compounds are present in a compound library.

In another aspect, the invention provides a compound comprising an amino acid motif having between 2 and 30 amino acids from Cdc25A and having a serine at a position corresponding to Serl7 or Serl 15 in full length Cdc25A in the design of an compound which is modelled to resemble the three dimensional structure, the steric size, and/or the charge distribution of the amino acid motif, the wherein the compound has the property of binding to cyclin B/Cdkl.

In another aspect, the invention provides a method of preparing a medicament for the treatment of cancer or a hyperproliferative disorder comprising combining a compostion which is capable of enhancing the interaction of Cdc25A and cyclin B/Cdkl, wherein the enhancement of the interaction promotes the phosphorylation of the Cdc25A on a serine at a position corresponding to Serl7 or Serl 15 in a full length Cdc25A, with a pharmaceutically acceptable carrier.

Preferably, the enhancement of the interaction additionally promotes the increase in the level of phosphorylated Cdc25A, or the interaction takes place in vivo and whereby the interaction additionally promotes transition from G2 phase to M phase.

In another embodiment, the invention provides a method wherein the composition comprises:

(a) a protein which has at least 80% sequence identity with a corresponding sequence of Cdc25A, the protein including a serine residue at a position corresponding to amino acid Serl7 or Serl 15 in Cdc25A, wherein the serine residue is phosphorylated; or,

(b) a derivative of protein (a); or

(c) a substance which is protein (a) or derivative (b) linked to a coupling partner.

Preferably, the composition comprises a proteasome inhibitor, such as LLnL or a functional derivative thereof, or the composition comprises caffeine or a functional derivative thereof. h another aspect, the invention provides a method for raising antibodies capable of specifically binding to phosphorylated Cdc25A,comprising use of a composition which is:

(a) a peptide fragment of between 5 and 30 amino acids which has at least 80% sequence identity with a corresponding sequence of Cdc25A, the fragment including a phosphorylated serine residue at a position corresponding to amino acid Serl7 or Serl 15 in Cdc25A; or,

(b) a derivative of peptide fragment (a); or

(c) a substance which is peptide fragment (a) or derivative (b) linked to an immunogenic carrier.

Alternatively, the invention also provides a method for raising antibodies capable of specifically binding to Cdc25A, as described above, but wherein the phosphorylated Cdc25A is phosphorylated on residues other than Serl7 or Serl 15 (particularly wherein it is phosphorylated on Serl 23 or Ser262 or Ser292 or Ser504), or wherein the Cdc25A is dephosphorylated.

In one embodiment, the method further comprises detection of the presence of Cdc25A, wherein the Cdc25A is phosphorylated at either Serl7 or Serl 15, whereby the detection comprises:

(a) binding of the antibodies substantially to the Cdc25A phosphorylated at either Serl7 or Serl l5;

(b) detecting the presence of the bound antibodies using

(i) size separation methods; or

(ii) any one of the following: chemiluminescence, enzymatic, immunological, or radiological methods.

In another embodiment, the peptide fragment further comprises between 7 and 15 amino acids which has at least 90% sequence identity with a corresponding sequence of Cdc25A. '

In another aspect, the invention provides a method for the preparation of a medicament for the treatment of cancer or a hyperproliferative disorder, comprising combining a composition which is capable of enhancing the interaction of cyclin B/Cdkl kinase and Cdc25A, wherein the enhancement of the interaction promotes the phosphorylation of the Cdc25A, with a pharmaceutically acceptable carrier. Preferably, the phosphorylation of the Cdc25A takes place on amino acid residues Serl7 or Serl 15, or the enhancement of the interaction additionally promotes the transition from G2 phase into M phase.

In another aspect, the invention provides a method of identification of patients having a functional Cdc25A phosphorylation-cyclin B/Cdkl kinase pathway in cancer cells comprising measuring the presence or absence of Cdc25A phosphorylation on Serl7 or Serl 15 following treatment of the cells with chemotherapy or radiotherapy.

Preferably, the chemotherapy includes a proteasome inhibitor, such as LLnL or a functional derivative thereof, or it includes caffeine or a functional derivative thereof.

Alternatively, the chemotherapy includes cyclin B/Cdkl kinase or a functional derivative thereof; Cdc25A or a functional derivative thereof; or the administration of a DNA topoisomerase toxin, such as an etoposide, an anthracycline, an epipodophyllotoxine or a camptothecin derivative.

In one embodiment, the radiotherapy includes the use of γ-radiation.

In another aspect, the invention provides a diagnostic kit for the identification of patients expressing Cdc25A, wherein the Cdc25A is phosphorylated on Serl 7 or Serl 15 in cancer cells comprising an antibody against phosphorylated Cdc25A.

In a preferred embodiment, the antibody is raised against a composition comprising a protein having at least 80% sequence identity with a corresponding sequence Cdc25A, the protein including serine at a position corresponding to amino acid Ser 17 or Serl 15 in Cdc25A.

i another aspect, the invention provides a method of screening a patient population by determining the level of Cdc25A phosphorylation in cancer cells derived from each patient comprising:

(a) providing a sample comprising tissue material or cells from a tumor;

(b) preparing the sample to obtain an appropriate tissue extract;

(c) optionally further treating the sample extract by one or more purification processes;

(d) contacting the tissue preparation with an antibody against phosphorylated Cdc25A to produce a Cdc25A-antibody primary complex;

(e) contacting the complex with a secondary antibody containing a specific label or reporter group to enable determination of the amount of Cdc25A present; and

(f) either

(i) comparing the amount of Cdc25 A with a standard sample to determine whether it is more or less than the standard amount; or (ii) comparing the level of activity of Cdc25 A with that of wild-type Cdc25A, wherein the wild-type Cdc25A has been phosphorylated on Serl 7 or Serl 15 or both, in a standard protein activity assay to determine whether it is more or less than the standard amount.

In one embodiment, the level of Cdc25A is determined using the Western blot method or ELISA. In another embodiment, the activity of Cdc25A may be measured as a function of its ability to activate Cdkl, preferably by dephosphorylation, particularly dephosphorylation of Thrl4 and/or Tyrl5.

In another aspect, the invention provides a method of screening a patient population by determining the presence of phosphorylated Cdc25A in cancer cells derived from each patient comprising:

(a) providing a sample comprising tissue material or cells from a tumor;

(b) preparing the sample to obtain an appropriate tissue extract under conditions selected to inhibit phosphorylation and dephosphorylation;

(c) contacting the tissue preparation with an antibody against Cdc25A to produce a Cdc5A-antibody complex;

(d) isolating the Cdc25A under conditions selected to inhibit phosphorylation or dephosphorylation;

(e) simultaneously subjecting to a size separation method under conditions selected to inhibit phosphorylation or dephosphorylation:

(i) the Cdc25A;

(ii) at least one Cdc25A standard comprising phosphorylated or non- phosphorylated Cdc25A;

(f) detecting the presence of the Cdc25A and the Cdc25A standard; and (g) observing the relative migration of the Cdc25A as compared to the Cdc25A standard, whereby phosphorylated Cdc25A migrates more slowly than non- phosphorylated Cdc24A.

In preferred embodiments, the size separation method of step (e) comprises a gel electrophoresis method, or step (f) detecting the presence of the Cdc25A and the Cdc25A standard comprises Western blotting.

In additional preferred embodiments, the chemotherapy may include an inhibitor of Weel or an inhibitor of Mytl, or the composition may comprise an inhibitor of Weel or an inhibitor of Mytl.

In additional preferred embodiments, the method for identifying compounds further comprises use of low throughput or high throughput techniques to measure the transition from G2 phase into M phase. Preferably, these techniques include microscopic techniques or flow cytometry.

In another aspect, the invention provides a method of sensitizing a patient for chemotherapy or radiotherapy or antisense therapy comprising administering at least one drug capable of promoting the Cdc25A-cyclin B/Cdkl pathway. For example, the antisense therapy may be used to decrease expression of F-box protein. In one embodiment, the drug is a peptide or peptide mimetic.

EXAMPLES 1 THROUGH 5

I. Materials and Methods

Cell culture.

Derivatives of the U-2-OS/TA cell line (Lukas et al., 1999) expressing HA-tagged alleles of Cdc25A in a tetracycline-dependent manner were generated and induced as described (Mailand et al., 2000). U-2-OS T-Rex cells (Invitrogen) were treated according to the manufacturer's instructions. Synchronisation of cells in early S phase by a double-thymidine block was described (Clute and Pines, 1999). Pure fractions of mitotic cells were obtained by shaking off the rounded cells following 12 h treatment with Nocodazole (40 ng/ml; Sigma). The Cdk inhibitor Roscovitine (25 μM; Calbiochem), the proteasome inhibitor LLnL (25 μM; Sigma), or the topoisomerase II poison Etoposide (0.5 μg/ml; Sigma) were added to culture medium for the time indicated in Figure legends. For estimation of the Cdc25A protein half- life, the cultures were either supplemented with cycloheximide (25 μg/ml) for the time indicated in figure legends, or the cells were metabolically labeled with [^3SS]methionine and subjected for the pulse-chase analysis as described (Lukas et al., 1995).

Plasmids, mutagenesis and protein purification.

Human Cdc25A cDNA was tagged at the amino terminus with the Haemagglutinin (HA) epitope and subcloned into the pBI tetracycline-responsive plasmid (Clontech). Phosphatase- dead (C430S) and A A (S17A/S115A) mutants were generated using the QuikChange Site- Directed Mutagenesis Kit (Stratagene). For tetracycline-inducible expression in U-2-OS T-Rex cells, HA-tagged Cdc25A alleles were subcloned into the pcDNA4/TO plasmid (Invitrogen). GST-Cdc25A(10-23) and GST-Cdc25A(108-121) fusion proteins were purified from Escherichia coli using standard procedures.

RT-PCR.

Total RNA was isolated using a Triazol reagent kit (Gibco-BRL). Conditions for reverse transcription (RT) and PCR amplification of Cdc25A and the porphobilinogen aminase (PBGD) housekeeping gene were described (Santoni-Rugiu et al., 2000). For RT-PCR amplification of Cdc25C mRNA, the following primers were used; 5'- ATGTCTACGGAACTCTTCTCA-3' (forward) and 5'-TGGAACAGTAGTAATGGGACT- 3' (reverse).

Immunochemical techniques.

Mouse monoclonal antibodies to Cdc25A (DCS-122, DCS-124), Cdc25B (DCS-162, DCS- 164), and Cdc25C (DCS- 193) were described (Mailand et al., 2000). Other immunoreagents included mouse monoclonal antibodies to Cdc25A (F-6; Santa Cruz), Cyclin B (C23420; Transduction Laboratories), Cdc27 (SC-9972; Santa Cruz), and HA-tag (12CA5; Lukas et al., 1997), rabbit serum (PC25) to Cdkl (Calbiochem), and a phospho-specific antibody (06-570) to histone H3 (Upstate Biotechnology). Immunoprecipitation, immunoblotting (Lukas et al., 1997) and Cdc25 phosphatase assays and measurement of Cyclin B-Cdkl kinase activity (Mailand et al., 2000) were described. For immunodepletion of Cdc25s, whole-cell lysates of asynchronous U-2-OS/TA cells in kinase assay buffer (50 mM HEPES, pH 7.5; 10 mM MgC12; 5 mM EGTA) were subjected to three successive immunoprecipitations with Cdc25 antibodies or pre-immune mouse serum. For in vitro dephosphorylation assays, λ-phosphatase (New England Biolabs) was used as recommended by the manufacturer. Cdc25A ubiquitination assay was determined as described (Mailand et al., 2000).

Flow cytometry.

DNA distribution of propidium iodide (PI)-stained cell nuclei was determined by the FACSCalibur flow cytometer (Becton Dickinson). Identification of mitotic cells was done by staining of chromosomal DNA by PI combined with immunofluorescent detection of phosphorylated histone H3 (Xu et al., 2001). Briefly, fixed cells were washed with PBS, suspended in 1 ml of 0.25% Triton X-100 in PBS, and incubated on ice for 15 min. After centrifugation, cells were resuspended in PBS with 1% bovine serum albumin (BSA) and 0.75 μg of phospho-histone H3 antibody and incubated for 3 h at room temperature. After rinsing with 1% BSA in PBS, cells were incubated with FITC-conjugated goat anti-rabbit IgG antibody (Jackson ImmunoResearch Laboratories, diluted 1:30) for 30 min in the dark, then stained with PI and analysed by flow cytometry.

Identification of phosphorylation sites by mass spectrometry.

Protein bands excised from gels were reduced, alkylated and digested with trypsin (Shevchenko et al., 1996). MALDI peptide maps were recorded on Reflex m mass spectrometer (Bruker-Daltonics) using 1-2% of the peptide mixtures. The remaining samples were desalted and eluted into nanoelectrospray (Wilm and Mann, 1996) needles using a combination of POROS R2 and POROS R3 microcolumns (PerSeptive Biosystems). Electrospray analysis was performed on a quadrupole-time-of-flight mass spectrometer (Shevchenko et al, 1997) QSTAR (PE-Sciex). Data interpretation and database searches were done using the Protein and Peptide Software Suite from MDS Proteomics (Odense, Denmark).

II. Results

A. Example 1: Cdc25A is stabilised in mitosis.

To elucidate mechanisms that determine the abundance of Cdc25A, the stability of the endogenous protein was measured in synchronised U-2-OS cells. Short inhibition of protein synthesis by cycloheximid revealed that in late Gl, S, and G2 phases, Cdc25A was degraded with an estimated half-life around 20 min (Fig.1 A). In contrast, Cdc25A became remarkably stabilised in purified mitotic cells synchronised by the microtubule-depolymerizing drug nocodazole (Fig. 1A), as judged from the extended half-life of well over 2 hours (Fig. IB). Cdc25C, on the other hand, remained stable under all conditions (Fig. IB). The overall protein turnover of Cdc25A in asynchronous U-2-OS cells measured by pulse-chase of the immunopurified radioactively-labelled protein and that obtained by inhibition of translation by cycloheximide revealed essentially identical values (half-life between 20-30 min) (Fig. 1C), thereby validating the latter approach employed in the rest of the study mainly to avoid unnecessary damage of fragile mitotic cells by procedures inevitably associated with radioisotope labeling. Since Cdc25A has been regarded as a Gl/S regulator, its accumulation and stabilisation in mitosis was interesting.

First, the concern that the mitotic accumulation of Cdc25A reflected increased transcription was excluded. In fact, a quantitative RT-PCR analysis showed that the Cdc25A (but not Cdc25C) mRNA was downregulated in nocodazole-arrested cells (Fig. ID), consistent with Cdc25 A gene expression being positively regulated by E2F, and with E2F silencing upon S-phase exit. Second, while treatment of asynchronous U-2-OS cells with a proteasome inhibitor stabilised the endogenous Cdc25A protein, it did not have any effect on Cdc25A in nocodazole-arrested cells (Fig. IE), suggesting that the mitotic form of Cdc25A is already fully stable. Finally, it was found that the Cdc25A protein was highly elevated also in cells naturally progressing through mitosis (Fig. IF), eliminating the possibility that the observed stabilisation could be a consequence of the spindle assembly checkpoint or any side effects elicited by nocodazole. In conclusion, the accumulation of human Cdc25A in mitosis occurs as a result of its protein stabilisation.

The next step was to determine whether the stabilised form of Cdc25A retains its activity. Indeed, in vitro phosphatase assays revealed that Cdc25A immunoprecipitated from mitotic cells was highly active, as was the known regulator of mitosis, Cdc25C (Fig.lG). Like Cdc25C, the mitotic Cdc25A was modified, as indicated by its retarded electrophoretic migration (Fig.lB,E-H). Treatment of immunopurified Cdc25A and C with λphosphatase reverted their migration back to the pattern seen in interphase cells (Fig.lG), indicating that their M-phase shifts were due to phosphorylation. Despite these similarities, at least three pieces of evidence suggest that the regulation of Cdc25A and C in mitosis profoundly differ. First, while the activity of Cdc25C was strictly associated with its shifted, phosphorylated form, Cdc25 A was active already during the interphase. Second, dephosphorylation by the λ phosphatase completely inhibited Cdc25C, while it had little effect on the activity of the mitotic Cdc25A (Fig. IG). Third, physiological dephosphorylation upon exit from mitosis correlated with gradual and complete destruction of the Cdc25A protein but did not alter the abundance of Cdc25C (Fig.lH). Together, these data suggest that in addition to activating Cdc25C the mitosis-specific phosphorylation is instrumental for building up the total activity of the Cdc25A phosphatase through its stabilisation.

B. Example 2: Stabilisation of Cdc25A in mitosis reflects cyclin B/Cdkl -mediated phosphorylation of Serl7 and Serll5.

The mobility shift and the appearance of stable Cdc25A coincided with the peak of activity of cyclin B/Cdkl, the main mitosis-promoting kinase (Fig.lG). Two additional results supported the link between cyclin B/Cdkl and phosphorylation-dependent stabilisation of Cdc25A. First, immunopurified cyclin B/Cdkl strongly phosphorylated GST-Cdc25A, resulting in a mobility shift similar to that observed on endogenous Cdc25A in mitosis (Fig.2A). This was completely reverted by Roscovitine, an inhibitor of Cdk2 and Cdkl (Fig.2A). Second, if the stability of the mitotic Cdc25A was dependent on CDK-mediated phosphorylation, inhibition of Cdkl should destabilise Cdc25A. Indeed, treatment of nocodazole-arrested cells with Roscovitine reduced the mitosis-specific mobility shift of Cdc25A and led to a rapid disappearance of the endogenous Cdc25A protein with the kinetics closely following dephosphorylation of known targets of cyclin B/Cdkl such as Cdc27 (Fig.2B). The major target of Roscovitine under these conditions must have been cyclin B/Cdkl because the other plausible candidates, cyclin A/Cdk2 or cyclin A/Cdkl are absent in nocodazole-arrested cells due to cyclin A degradation. To identify the residues of Cdc25A targeted by cyclin B/Cdkl in vivo, the Cdc25A protein isolated either from asynchronous or nocodazole-arrested cells was subjected to mass spectrometry. To obtain enough material for this analysis, a U-2-OS cell line conditionally expressing ectopic Cdc25A was employed. Although this technique may not necessarily identify all phosphorylations, two prominent targets, Serl7 and Serl 15 within the Cdc25A N- terminal regulatory domain, were specifically phosphorylated in mitotic but not interphase cells. The regions surrounding these serines are highly homologous in human, mouse and rat proteins (Fig.2C), suggesting that modification of S17/S115 by cyclin B/Cdkl could represent a conserved regulatory mechanism.

C. Example 3: Mutation of Serl7/Serll5 abolishes Cdc25A phosphorylation in vitro, and restores ubiquitylation and instability of the mitotic Cdc25A in vivo.

To test whether the identified serines could be phosphorylated in mitosis, GST-tagged fragments of Cdc25A containing Serl7 and Serl 15, respectively, were purified from bacteria. Equivalents of these fusion proteins where the serines were substituted by alanines were also generated and subjected to in vitro phosphorylation by cyclin B/Cdkl from mitotic cell extracts. While both fusion proteins containing the wild-type serines were efficiently phosphorylated in a CDK-dependent manner, the alanine mutants remained virtually unmodified (Fig.3A,B). Furthermore, analogous mutations in the context of the full-length GST-Cdc25A protein also impaired (although did not completely abolish) its phosphorylation by cyclin B/Cdkl in this in vitro kinase assay.

Next, both Serl 7 and Serl 15 in the context of the full-length Cdc25A were substituted with alanines and U-2-OS clones conditionally expressing the wild-type or the double-alanine (A/A) mutant of Cdc25 A in a tetracycline-dependent manner were generated. Upon induction in asynchronous cells, both forms of Cdc25A accumulated with a very similar kinetics (Fig.3C). Strikingly, when induced in nocodazole-arrested cells, the phosphorylation-deficient Cdc25A(A/A) mutant accumulated more slowly than the wild-type protein (Fig.3C), a difference abolished in the presence of the proteasome inhibitor LLnL (Fig.3C). These results are consistent with an interpretation that the cyclin B/Cdkl -mediated phosphorylation of S17/S115 is instrumental for stabilisation and accumulation of Cdc25A specifically in mitotic cells. This conclusion was also supported by direct protein stability estimation, as wild-type Cdc25A was stable in mitosis, while the Cdc25A(A/A) mutant was turned over rapidly (Fig. 3D).

Finally, if the lack of S17/S115 phosphorylation indeed induced destruction of Cdc25A in mitosis, it should be reflected by the increased propensity of the mutated protein to undergo polyubiquitylation, a prerequisite for targeting of many proteins by the proteasome. This was tested by an established in vivo assay, which showed that wild-type Cdc25A was ubiquitylated in asynchronous cells but not in mitosis (Fig.3E). In contrast, Cdc25A(A/A) became polyubiquitylated also in mitotic cells (Fig.3F). These data demonstrate that the mitotic phosphorylation of S17/S115 uncouples Cdc25A from the ubiquitin/proteasome-mediated degradation and leads to accumulation of an active Cdc25A phosphatase.

D. Example 4: Cdc25A activates cyclin B/Cdkl and is rate-limiting for G2/M transition.

It has been reported that the sharp increase of cyclin B/Cdkl activity early in mitosis and its subsequent maintenance is essential for proper execution of mitotic events. To address this issue, each member of the Cdc25 family (Fig.4A) was immunodepleted and the ability of the resulting cell extracts to activate cyclin B/Cdkl in vitro was assayed. Depletion of each individual Cdc25 phosphatase reduced the total cellular cyclin B/Cdkl -activating potential to about 50% (Fig.4B). Combined depletion of Cdc25B and C was more effective, yet a complete inhibition, mimicking the effect of the phosphatase inhibitor, sodium vanadate, required depletion of all three Cdc25s. Thus, Cdc25A, B and C jointly generate a cellular phosphatase pool required for full activation of cyclin B/Cdkl in mitosis. In addition, Cdc25A and cyclin B/Cdkl appear to stimulate each other in a positive feedback loop. Cyclin B/Cdkl enables Cdc25A accumulation in mitosis and the stabilised Cdc25A, in turn, helps maintain the cyclin B/Cdkl kinase in an active state, presumably until the destruction of cyclin B at the metaphase-anaphase transition.

Such results suggest that Cdc25A can activate cyclin B/Cdkl in vivo and thereby modulate G2/M progression. Indeed, conditionally elevated Cdc25A bound physically to cyclin B and Cdkl in vivo, and caused a strong increase of endogenous cyclin B/Cdkl kinase activity (Fig.4C). Consistent with activation of cyclin B/Cdkl, transient induction of wild-type Cdc25A accelerated entry into mitosis from G2 (Fig.4D), and induced premature mitosis in cells with incompletely replicated DNA (Fig.4D). Conversely, neutralisation of Cdc25A by conditional overexpression of its dominant-negative, phosphatase-dead mutant (Fig.4E) inhibited not only the S-phase entry as expected (Fig.4F), but delayed also the G2/M transition (Fig.4G). Remarkably, the G2/M delay occurred in spite of the fact that the activities of Cdc25B and Cdc25C in cells overexpressing the phosphatase-dead Cdc25A remained unchanged (Fig.4H). Thus, apart from its S phase-promoting effect, Cdc25A physically and functionally interacts with the main mitosis-promoting cyclin/CDK complex, generates a rate- limiting stimulus for G2/M transition, and the lack of its activity can delay completion of the cell division cycle. E. Example 5: Destruction of Cdc25A is required to prevent entry into mitosis in response to DNA damage.

The above data implicate Cdc25A as a potential target of the checkpoint pathways that delay entry into mitosis under various stress conditions. To test this possibility, U-2-OS cells were transiently treated with the topoisomerase II inhibitor etoposide, a drug known to generate chromosomal stress including DNA double-strand breaks, and elicit a robust G2 arrest. Exposure to etoposide caused rapid destruction of the Cdc25A protein and loss of its phosphatase activity. In contrast, the protein level and activity of Cdc25C remained unchanged (Fig.5A). Addition of either the proteasome inhibitor LLnL, or caffeine that inhibits the ATM/ATR kinases rescued the downregulation of Cdc25A in the etoposide-treated cells. Thus, similarly to cells exposed to genotoxic stress in late Gl or S phase, the G2 checkpoint was also accompanied by the accelerated proteolysis of Cdc25A.

To test whether the Cdc25A degradation was essential for the G2 checkpoint, the U-2- OS clones conditionally expressing various Cdc25s were exposed to etoposide. The parental U-2-OS/TA and its derivative expressing Cdc25C responded to etoposide by a robust cessation of mitotic entry (Fig.5B). In contrast, short pre-induction of Cdc25A to the levels sufficient to compensate its degradation caused partial but reproducible abrogation of the G2 checkpoint and resulted in reduced but steady progression of the damaged cells into mitosis (Fig.5B). Strikingly, the mitotic, cyclin B/Cdkl -phosphorylated form of Cdc25A remained stable even after exposure to relatively high doses of ionizing radiation (Fig.5C). This indicates that the stabilising phosphorylations (including those on Serl7/Serl 15 identified here) are dominant and sufficient to uncouple Cdc25 A from those regulatory mechanisms, which normally determine the basic interphase turnover as well as the accelerated proteolysis in response to DNA damage. Taken together, these data support the model of Cdc25A and its role in mitosis as depicted in Figs. 6A-6B.

The foregoing information can be summarized as follows:

DNA replication in higher eukaryotes requires activation of a Cdk2 kinase by Cdc25A, a labile phosphatase subject to further destabilisation upon genotoxic stress. A distinct, markedly stable form of Cdc25A plays a previously unrecognized role in mitosis. Mitotic stabilisation of Cdc25A reflects its phosphorylation on Serl7 and Serl 15 by cyclin B/Cdkl, modifications required to uncouple Cdc25A from its ubiquitin/proteasome-mediated turnover. In turn, mitotic Cdc25A binds and activates cyclin B/Cdkl, and accelerates cell division. DNA damage-induced G2 arrest, in contrast, is accompanied by proteasome- dependent destruction of Cdc25A, and ectopic Cdc25 A abrogates the G2 checkpoint. Thus, phosphorylation-mediated switches among three differentially stable forms ensure distinct thresholds, and thereby distinct roles of Cdc25 A in multiple cell cycle transitions and checkpoints.

The references mentioned herein are all expressly incorporated by reference.

The invention has been described in detail with reference to preferred embodiments thereof. However, it will be appreciated that those skilled in the art, upon consideration of the disclosure, may make modifications and improvements within the spirit and scope of the invention.

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Claims

CLAIMSWhat is claimed is:

1. A method for treating or preventing cancer or a hyperproliferative disorder comprising administering a composition which is capable of enhancing the interaction of Cdc25 A and cyclinB/Cdkl.

2. The method of claim 1, wherein the composition enhances the binding of Cdc25A to cyclin B/Cdkl or the phosphorylation of Cdc25 A by cyclin B/Cdkl .

3. The method of claim 1 or claim 2, wherein the hyperproliferative disorder is psoriasis, arteriogenesis, angiogenesis, inflammation, or non-malignant tumor.

4. The method of any one of claims 1 to 3, wherein the composition is administered in combination with chemotherapy or radiotherapy.

5. The method of claim 4, wherein the chemotherapy includes a proteasome inhibitor.

6. The method of claim 5, wherein the proteasome inhibitor comprises LLnL or a functional derivative thereof.

7. The method of claim 4, wherein the chemotherapy includes caffeine or a functional derivative thereof.

8. The method of claim 4, wherein the chemotherapy includes cyclin B/Cdkl kinase or a functional derivative thereof.

9. The method of claim 4, wherein the chemotherapy includes Cdc25 A that is phosphorylated on at least one of Serl7 or Serl 15, or a functional derivative thereof.

10. The method of any one of claims 4 to 9, wherein the chemotherapy includes the administration of a DNA topoisomerase toxin.

11. The method of claim 10, wherein the DNA topoisomerase toxin is an etoposide, an anthracycline, an epipodophyllotoxine or a camptothecin derivative.

12. The method of any one of claims 4 to 9, wherein the radiotherapy includes the use of γ-radiation.

13. The method of any one of the preceding claims, wherein the composition comprises a protein complex comprising:

(a) a first protein moiety comprising:

(i) a protein which has at least 80% sequence identity with a corresponding sequence of cyclin B; or

(ii) a derivative of protein (ai); or

(iii) a substance which is protein (ai) or derivative (aii) linked to a coupling partner; and

(b) a second protein moiety comprising:

(i) a protein which has at least 80% sequence identity with a corresponding sequence of Cdkl; or

(ii) a derivative of protein (bi); or

(iii) a substance which is protein (bi) or derivative (bii) linked to a coupling partner.

14. The method of any one of the above claims, wherein the composition comprises:

(a) a protein which has at least 80% sequence identity with a corresponding sequence of Cdc25A, the protein including a phosphorylated serine residue at a position corresponding to amino acid Serl 7 or Ser 115 in Cdc25A; or,

(b) a derivative of protein (a); or

(c) a substance which is protein (a) or derivative (b) linked to a coupling partner.

15. The method of any one of the above claims, wherein the composition comprises cyclin B/Cdkl or a functional derivative thereof capable of phosphorylating Cdc25A at Serl7 or Serl 15.

16. The method of any one of the above claims, wherein the composition comprises Cdc25A or a functional derivative thereof, capable of enhancing the activity of cyclin B/Cdkl kinase.

17. The method of any one of the above claims wherein:

(a) the composition comprises Cdc25A or a functional derivative thereof, including a phosphorylated serine residue at a position corresponding to amino acid Serl 7 or Ser 115 in Cdc25A; and

(b) the composition is capable of enhancing the activity of cyclin B/Ckdl kinase.

18. The method of any one of claims 13 to 17, wherein

(a) the cyclin B comprises a mammalian cyclin B; or

(b) the Cdkl comprises a mammalian Cdkl.

19. The method of any one of claims 14 to 17, wherein the Cdc25A comprises as mammalian Cdc25A.

20. The method of any one of claims 1 to 12, wherein the composition comprises an inhibitor of proteasome degradation.

21. The method of claim 20, wherein the inhibitor comprises LLnL or a functional derivative thereof.

22. The method of any one of claims 1 to 12, wherein the composition comprises caffeine or a functional derivative thereof.

23. The method of any one of the previous claims, wherein the method further comprises administering the composition to a mammal in an amount sufficient to increase the level of Cdc25A by at least 10% as determined by a standard protein assay.

24. A composition having the property of promoting the phosphorylation of Cdc25A or a derivative thereof, wherein the Cdc25A or the derivative thereof, includes a serine residue at a position corresponding to amino acid Serl 7 or Serl 15 in Cdc25A, and wherein the composition comprises a protein complex comprising:

(a) a first protein moiety comprising:

(i) a protein which has at least 80% sequence identity with a corresponding sequence of cyclin B; or

(ii) a derivative of protein (ai); or

(iii) a substance which is protein (ai) or derivative (aii) linked to a coupling partner; and

(b) a second protein moiety comprising:

(i) a protein which has at least 80% sequence identity with a corresponding sequence of Cdkl; or

(ii) a derivative of protein (bi); or

(iii) a substance which is protein (bi) or derivative (bii) linked to a coupling partner.

25. A composition having the property of enhancing the activity of cyclin B/Cdkl and promoting the phosphorylation of Cdc25 A by the cyclin B/Cdkl , the composition comprising:

(a) a protein which has at least 80% sequence identity with a corresponding sequence of Cdc25A, the protein including a serine residue at a position corresponding to amino acid Serl 7 or Serl 15 in Cdc25A; or,

(b) a derivative of protein (a); or

(c) a substance which is protein (a) or derivative (b) linked to a coupling partner.

26. An isolated nucleic acid molecule encoding the composition of claim 24 or claim 25.

27. An expression vector comprising the nucleic acid of claim 26, operably linked to sequences to direct its expression.

28. A host cell transformed with the expression vector of claim 27.

29. A method of producing the composition of claim 25, the method comprising culturing the host cells of claim 28 and isolating the composition thus produced.

30. The composition of claim 25 for use in a method of medical treatment.

31. A pharmaceutical composition comprising the composition of claim 25.

32. A method of using the composition of claim 25 for identifying (i) binding partners of the composition or (ii) compounds having the property of enhancing the activity of cyclin B/Cdkl kinase and promoting the phosphorylation of Cdc25A.

33. A method of identifying compounds capable of modulating the interaction of Cdc25A and cyclin B/Cdkl kinase, the method comprising:

(a) contacting (i) a composition comprising Cdc25A or a fragment or variant thereof, (ii) a composition comprising cyclin B/Cdkl kinase or a fragment or variant thereof and (iii) a candidate compound, under conditions wherein, in the absence of the candidate compound, the compositions interact; and

(b) determining the interaction between the compositions to identify whether the candidate compound modulates the interaction.

34. The method of claim 33, wherein the interaction determined in step (b) is the binding of Cdc25A by cyclin B/Cdkl kinase.

35. The method of claim 33, wherein interaction determined in step (b) is the phosphorylation of Cdc25A by cyclin B/Cdkl kinase, or the presence or amount of Cdc25A present in a cell based assay.

36. The method of any one of claims 33 to 35, wherein the compound capable of modulating the interaction of Cdc25A and cyclin B/Cdkl kinase is capable of enhancing the interaction or the phosphorylation of Cdc25A by cyclin B/Cdkl kinase.

37. The method of any one of claims 33 to 36, wherein the Cdc25A is fusion of GST and a fragment of Cdc25A comprising an amino acid sequence corresponding to the Ser 17 or Serl 15 of full length Cdc25A.

38. The method of any one of claims 33 to 37, comprising determining the modulation of the interaction of Cdc25A and cyclin B/Cdkl by measuring the phosphorylation of the Cdc25A peptide.

39. The method of claim 38, wherein the phosphorylation of Cdc25A is measured by the incorporation of radioactive phosphate into the Cdc25A peptide.

40. The method of claim 38, wherein the phosphorylation of Cdc25A is determined using an antibody capable of specifically binding to phosphorylated Cdc25 A peptide.

41. The method of any one of claims 33 to 40, further comprising testing a candidate compound identified in step (b) to determine whether it is capable of promoting transition from G2 phase into M phase in a population of cells.

42. A method of identifying binding partners of a composition having the property of enhancing the activity of cyclin B/Cdkl kinase and promoting the phosphorylation of Cdc25A by the cyclin B/Cdkl kinase, the composition comprising a protein having at least 80% sequence identity with a corresponding sequence of Cdc25A, the protein including serine at a position corresponding to amino acid Serl7 or Serl 15 in Cdc25A, the method comprising contacting the composition and a candidate compound and determining whether the candidate compound has the property of binding to the composition.

43. The method of claim 42, further comprising testing the compounds which bind to Cdc25A for activity in promoting the phosphorylation of Cdc25A by cyclin B/Cdkl kinase.

44. The method of claim 42 or claim 43, further comprising testing the candidate compound to determine whether it is capable of promoting transition from G2 phase into M phase in a population of cells.

45. The method of any one of claims 33 to 44, wherein a plurality of candidate compounds are contacted with the compositions.

46. The method of claim 45, wherein the plurality of compounds are present in a compound library.

47. A compound comprising an amino acid motif having between 2 and 30 amino acids from Cdc25A and having a serine at a position corresponding to Serl7 or Serl 15 in full length Cdc25A in the design of an compound which is modelled to resemble the three dimensional structure, the steric size, and/or the charge distribution of the amino acid motif, the wherein the compound has the property of binding to cyclin B/Cdkl .

48. A method of preparing a medicament for the treatment of cancer or a hyperproliferative disorder comprising combining a composition which is capable of enhancing the interaction of Cdc25A and cyclin B/Cdkl, wherein the enhancement of the interaction promotes the phosphorylation of the Cdc25A on a serine at a position corresponding to Serl 7 or Serl 15 in a full length Cdc25A, with a pharmaceutically acceptable carrier.

49. The method of claim 48, wherein the enhancement of the interaction additionally promotes the increase in the level of phosphorylated Cdc25A.

50. The method of claim 48, whereby the interaction takes place in vivo and whereby the interaction additionally promotes transition from G2 phase to M phase.

51. The method of any one of claims 48 to 50, wherein the composition comprises:

(a) a protein which has at least 80% sequence identity with a corresponding sequence of Cdc25 A, the protein including a serine residue at a position corresponding to amino acid Serl7 or Serl 15 in Cdc25A, wherein the serine residue is phosphorylated; or,

(b) a derivative of protein (a); or

(c) a substance which is protein (a) or derivative (b) linked to a coupling partner.

52. The method of any one of claims 48 to 50, wherein the composition comprises a proteasome inhibitor.

53. The method of claim 52, wherein the composition comprises LLnL or a functional derivative thereof.

54. The method of any one of claims 48 to 50, wherein the composition comprises caffeine or a functional derivative thereof.

55. A method for raising antibodies capable of specifically binding to phosphorylated Cdc25A, comprising use of a composition which is: (a) a peptide fragment of between 5 and 30 amino acids which has at least 80% sequence identity with a corresponding sequence of Cdc25A, the fragment including a phosphorylated serine residue at a position corresponding to amino acid Serl7 or Serl 15 in Cdc25A; or,

(b) a derivative of peptide fragment (a); or

(c) a substance which is peptide fragment (a) or derivative (b) linked to an immunogenic carrier.

56. The method of claim 55, further comprising detection of the presence of Cdc25A, wherein the Cdc25A is phosphorylated at either Serl7 or Serl 15, whereby the detection comprises:

(a) binding of the antibodies substantially to the Cdc25A phosphorylated at either Serl7 or Serl l5;

(b) detecting the presence of the bound antibodies using

(i) size separation methods; or

(ii) any one of the following: chemiluminescence, enzymatic, immunological, or radiological methods.

57. The peptide fragment of claim 51 or claim 56, further comprising between 7 and 15 amino acids which has at least 90% sequence identity with a corresponding sequence of Cdc25A.

58. A method for the preparation of a medicament for the treatment of cancer or a hyperproliferative disorder, comprising combining a composition which is capable of enhancing the interaction of cyclin B/Cdkl kinase and Cdc25A, wherein the enhancement of the interaction promotes the phosphorylation of the Cdc25A, with a pharmaceutically acceptable carrier.

59. The method of claim 58, wherein the phosphorylation of the Cdc25A takes place on amino acid residues Serl7 or Serl 15.

60. The method of claim 58, wherein the enhancement of the interaction additionally promotes the transition from G2 phase into M phase.

61. A method of identification of patients having a functional Cdc25 A phosphorylation- cyclin B/Cdkl kinase pathway in cancer cells comprising measuring the presence or absence of Cdc25A phosphorylation on Serl 7 or Serl 15 following treatment of the cells with chemotherapy or radiotherapy.

62. The method of claim 61 , wherein the chemotherapy includes a proteasome inhibitor.

63. The method of claim 62, wherein the proteasome inhibitor comprises LLnL or a functional derivative thereof.

64. The method of claim 61 , wherein the chemotherapy includes caffeine or a functional derivative thereof.

65. The method of claim 61 , wherein the chemotherapy includes cyclin B/Cdkl kinase or a functional derivative thereof.

66. The method of claim 61, wherein the chemotherapy includes Cdc25A or a functional derivative thereof.

67. The method of any one of claims 61 to 66, wherein the chemotherapy includes the administration of a DNA topoisomerase toxin.

68. The method of any one of claims 61 to 66, wherein the DNA topoisomerase toxin is an etoposide, an anthracycline, an epipodophyllotoxine or a camptothecin derivative.

69. The method of claim 61 , wherein the radiotherapy includes the use of γ-radiation.

70. A diagnostic kit for the identification of patients expressing Cdc25A, wherein the Cdc25A is phosphorylated on Serl7 or Serl 15 in cancer cells comprising an antibody against phosphorylated Cdc25A.

71. The diagnostic kit of claim 70, wherein the antibody is raised against a composition of claim 42.

72. A method of screening a patient population by determining the level of Cdc25 A phosphorylation in cancer cells derived from each patient comprising:

(a) providing a sample comprising tissue material or cells from a tumor;

(b) preparing the sample to obtain an appropriate tissue extract;

(c) optionally further treating the sample extract by one or more purification processes;

(d) contacting the tissue preparation with an antibody against phosphorylated Cdc25A to produce a Cdc25A-antibody primary complex;

(e) contacting the complex with a secondary antibody containing a specific label or reporter group to enable determination of the amount of Cdc25A present; and

(f) either

(i) comparing the amount of Cdc25 A with a standard sample to determine whether it is more or less than the standard amount; or (iϊ) comparing the level of activity of Cdc25 A with that of wild-type Cdc25A, wherein the wild-type Cdc25A has been phosphorylated on Serl 7 or Serl 15 or both, in a standard protein activity assay to determine whether it is more or less than the standard amount.

73. The method of claim 72 wherein the level of Cdc25 A is determined using the Western blot method or ELISA.

74. A method of screening a patient population by determining the presence of phosphorylated Cdc25A in cancer cells derived from each patient comprising:

(a) providing a sample comprising tissue material or cells from a tumor;

(b) preparing the sample to obtain an appropriate tissue extract under conditions selected to inhibit phosphorylation and dephosphorylation;

(c) contacting the tissue preparation with an antibody against Cdc25A to produce a Cdc5A-antibody complex;

(d) isolating the Cdc25A under conditions selected to inhibit phosphorylation or dephosphorylation;

(e) simultaneously subjecting to a size separation method under conditions selected to inhibit phosphorylation or dephosphorylation: (i) the Cdc25A;

(ii) at least one Cdc25A standard comprising phosphorylated or non- phosphorylated Cdc25A;

(f) detecting the presence of the Cdc25A and the Cdc25A standard; and

(g) observing the relative migration of the Cdc25A as compared to the Cdc25A standard, whereby phosphorylated Cdc25 A migrates more slowly than non- phosphorylated Cdc24A.

75. The method of claim 74, wherein the size separation method of step (e) comprises a gel electrophoresis method.

76. The method of claim 74, wherein step (f) detecting the presence of the Cdc25A and the Cdc25A standard comprises Western blotting.

77. The method of claim 4 or claim 61 , wherein the chemotherapy includes an inhibitor of Weel.

78. The method of claim 4 or claim 61, wherein the chemotherapy includes an inhibitor of Mytl.

79. The method of any one of claims 1 to 12 or of any one of claims 48-50, wherein the composition comprises an inhibitor of Weel.

80. The method of any one of claims 1 to 12 or of any one of claims 48-50, wherein the composition comprises an inhibitor of Mytl.

81. The method of claim 41 , claim 44, or claim 50, further comprising use of low throughput or high throughput techniques to measure the transition from G2 phase into M phase.

82. The method of claim 41, claim 44, or claim 50, further comprising use of microscopic techniques to measure the transition from G2 phase into M phase.

83. The method of claim 41, claim 44, or claim 50, further comprising use of flow cytometry to measure the transition from G2 phase into M phase.

84. The method of claim 23, wherein the Cdc25 is phosphorylated on either Serl7 or Serl 15.

85. A method of sensitizing a patient for chemotherapy or radiotherapy or antisense therapy comprising administering at least one drug capable of promoting the Cdc25A-cyclin B/Cdkl pathway.

86. The method of claim 85, wherein the drug is a peptide or peptide mimetic.

87. The method of claim 72, wherein the level of activity of Cdc25 A is measured as a function of activation of Cdkl .