AU2003235055B2 - Human checkpoint kinase, HCDS1, compositions and methods - Google Patents

Description

AUSTRALIA

Patents Act 1990 COMPLETE SPECIFICATION STANDARD PATENT Applicant(s): THE SCRIPPS RESEARCH INSTITUTE Invention Title: HUMAN CHECKPOINT KINASE, HCDS1, COMPOSITIONS AND

METHODS

The following statement is a full description of this invention, including the best method of performing it known to us: HUMAN CHECKPOINT KINASE, HCDSI COMPOSITIONS AND METHODS BACKGROUND OF THE INVENTION The integrity of the genome is of prime importance to a dividing cell. In response to DNA damage, eukaryotic cells rely upon a complex system of checkpoint controls to delay cell-cycle progression. The normal eukaryotic cell-cycle is divided into 4 phases (sequentially Gl, S, G2, M) which correlate with distinct cell morphology and biochemical activity, and cells withdrawn from the cell-cycle are said to be in GO, or non-cycling state. When cells within the cell-cycle are.actively replicating, duplication of DNA occurs in the S phase, and active division of the cell occurs in M phase. See generally Benjamin Lewin, GENES VI (Oxford University Press, Oxford, GB, Chapter 36, 1997). DNA is organized in the eukaryotic cell into successively higher levels of organization that result in the formation of chromosomes.

Non-sex chromosomes are normally present in pairs, and during cell division, the DNA of each chromosome replicates resulting in paired chromatids. (See generally Benjamin Lewin, GENES VI (Oxford University Press, Oxford, GB, Chapter 1997).

Checkpoint delays provide time for repair of damaged DNA prior to its replication in S-phase and prior to segregation of chromatids in M-phase (Hartwell and Weinert, 1989, Science. 246: 629-634). In many cases the DNA-damage response pathways cause arrest by inhibiting the activity of the cyclin-dependent kinases (Elledge, 1997, Science. 274: 1664-1671). In human cells the DNA-damage induced G2 delay is largely dependent on inhibitory phosphorylation of Cdc2 (Blasina et al., 1997, Mol. Cell Biol., 8: 1-11; Jin et al., 1996, J. Cell Biol.. 134: 963-970), and is therefore likely to result from a change in the activity of the opposing kinases and phosphatases that act on Cdc2. However, evidence that the activity of these enzymes is substantially altered in response to DNA damage is lacking (Poon et al., 1997, Cancer Res.. 57: 5168-5178).

Three distinct Cdc25 proteins are expressed in human cells. Cdc25A is specifically required for the GI-S transition (Hoffmann et al., 1994, EMBO 13: 4302-4310; Jinno et al., 1994, EMBO 13: 1549-1556), whereas Cdc25B and are required for the G2-M transition (Gabrielli et al., 1996, J. Cell Sci., 7: 1081-1093; Galaktionov et al., 1991, Cell. 67: 1181-1194; Millar et al., 1991, Proc.

Natl. Acad. Sci. USA, 88: 10500-10504; Nishijima et al., 1997, J. Cell Biol.. 138: 26/02 '07 MON 14:16 FAX 61299255911 GRIFFITH HACK [a005

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O 2 t 1105-1116). The exact contribution of Cdc25B and Cdc25C to M-phase progression is vnot known.

SMuch of our current knowledge about checkpoint control has been obtained from studies using budding (Saccharomyces cerevisiae) and fission I' 5 (Schizosaccharomyces pombe) yeast A number of reviews of our current o understanding of cell cycle checkpoints in yeast and higher eukaryotes have recently been published (Hartwell Kastan, 1994, Science 266; 1821-1828; Murray, 1994, Current Biology, 6: 872-876; Elledge, 1996, Science, 274: 1664-1672; Kaufmann o Paules, 1996, FASEB 10: 238-247). In the fission yeast six gene products, radl, rad3*, rad9, radl7, rad26 and hul* have been identified as components of both the DNA-damage dependent and DNA-replication dependent checkpoint pathways. In addition cdsl* has been identified as being required for the DNA-replication dependent checkpoint and rad27/chkl* has been identified as required for the DNAdamage dependent checkpoint in yeast.

Several of these genes have structural homologues in the budding yeast and further conservation across eukaryotes has recently been suggested with the cloning of two human homologues of S. pombe rad3*: ATM (ataxia telangiectasia mutated) (Savitsky et aL, 1995, Science. 268: 1749-1753) and ATR (ataxia telangiectasia and rad3 t related)(Bentley et al, 1996, EMBO 15: 6641-6651; Cimprich et al., 1996, Proc. Nati. Acad. Sci. USA. 93: 2850-2855) and of a human homologue of S. pombe radP*(Lieberman et aL, 1996, Proc. Natl. Acad. Sci. USA. 93; 13890-13885).

While much is known about yeast checkpoint proteins and genes, this knowledge is not fully predictive of the existence of corresponding human genes or proteins, dr their effector role in human cell-cycle control and regulation.

In order to develop new and more effective treatments and therapeutics for the amelioration of the effects of cancer, it is important to identify and characterize human checkpoint proteins and to identify mediators of their activity.

COMS ID No: SBMI-06388059 Received by IP Australia: Time 14:29 Date 2007-02-26 26/02 '07 MON 14:16 FAX 61299255911 GRIFFITH HACK S0oo6 0

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SSUIMMARY OF THE INVENTION In a first aspect, the invention provides a method of treating cancer or proliferative disease comprising administering a therapeutically effective amount s of a compound that is an inhibitor or activator of expression of the CDS1 human Scell cycle checkpoint pathway protein comprising the amino acid sequence i depicted in Figure 2 (SEQ ID NO: to a patient in need of such treatment, 0 wherein the compound is identified by a method which comprises contacting a C cell expressing the proteins of the CDS1 human cell cycle checkpoint pathway with said compound, and comparing the level of expression of the CDS1 human cell cycle checkpoint pathway protein of said cell against a cell which has not been in contact with said compound, wherein said compound increases or decreases expression of the CDS1 human cell cycle checkpoint pathway protein.

In one embodiment, the method further comprises administering a DNA damaging chemotherapeutic agent.

In one embodiment, the compound is administered by administering a pharmaceutical composition comprising the compound together with a pharmaceutically acceptable carrier, diluent or excipient therefor. The composition may further comprise a DNA damaging chemotherapeutic agent.

In a second aspect, the invention provides a method of treating cancer or proliferative disease comprising administering a therapeutically effective amount of a substance obtained by screening candidate substances in a method which comprises: providing a protein comprising the amino acid sequence of SEQ ID NO.: 2 or a functional equivalent, wherein said functional equivalent comprises conservative substitutions, deletions or insertions of the amino acid sequence of Figure 2 such that said protein comprises different amino acids than those of the amino acid sequence shown in Figure 2, yet which substitutions, deletions or insertions do not affect the activity of the protein having the sequence of Figur 2 COMS ID No: SBMI-06388059 Received by IP Australia: Time 14:29 Date 2007-02-26 26/02 '07 MON 14:16 FAX 61299255911 GRIFFITH HACK o007 0

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2a S(SEQ ID NO: as measured by the ability to interact in the human cell cycle checkpoint pathway, wherein the protein or equivalent of said protein exhibits a Va N kinase activity; contacting a substrate for said protein with the protein under conditions such tn 5 that the kinase will act upon the substrate; I bringing the protein and substrate into contact with a candidate substance; C, measuring the degree of increase or decrease in the kinase activity of the 0 protein; and selecting a candidate substance which provides a decrease or increase in said kinase activity to thereby obtain the substance.

In one embodiment, the method further comprises administering a DNA damaging chemotherapeutic agent.

In one embodiment, the substance is administered by administering a pharmaceutical composition comprising the substance together with a pharmaceutically acceptable carrier, diluent or excipient therefor. The composition may further comprise a DNA damaging chemotherapeutic agent.

In a third aspect, the invention provides a method of increasing susceptibility of cancer cells to chemotherapy and/or radiotherapy, which method comprises administering to a patient a therapeutically effective amount of a 2 substance obtained by screening candidate substances in a method which comprises: providing a protein comprising the amino acid sequence of SEQ ID NO.: 2 or a functional equivalent, wherein said functional equivalent comprises conservative substitutions, deletions or insertions of the amino acid sequence of Figure 2 such that said protein comprises different amino acids than those of the amino acid sequence shown in Figure 2, yet which substitutions, deletions or insertions do not affect the activity of the protein having the sequence of Figure 2 (SEQ ID NO: as measured by the ability to interact in the human cell cycle COMS ID No: SBMI-06388059 Received by IP Australia: Time 14:29 Date 2007-02-26 26/02 '07 MON 14:17 FAX 61299255911 GRIFFITH HACK 0 2C 0 g checkpoint pathway, wherein the protein or equivalent of said protein exhibits a o kinase activity; contacting a substrate for said protein with the protein under conditions such that the kinase will act upon the substrate; bringing the protein and substrate into contact with a candidate substance; S(d) measuring the degree of increase or decrease in the kinase activity of the CC) protein; and S(e) selecting a candidate substance which provides a decrease or increase in said N kinase activity to thereby obtain the substance.

In a fourth aspect, the invention provides a method of treating cancer or proliferative disease in a patient comprising administering a therapeutically effective amount of a compound capable of modifying the activity of CDS1 human cell cycle checkpoint pathway protein comprising the amino acid sequence depicted in Figure 2 (SEQ ID NO: to a patient in need of such treatment, wherein the compound is identified by a method which comprises combining Cdc25 protein with human hCDS 1 protein in the presence of ATP and the said compound, and detecting Cdc25 phosphatase activity to thereby obtain said compound.

In one embodiment, the method further comprises administering a DNA 2 0 damaging chemotherapeutic agent.

In one embodiment, the compound is administered by administering a pharmaceutical composition comprising the compound together with a pharmaceutically acceptable carrier, diluent or excipient therefor. The composition may further comprise a DNA damaging chemotherapeutic agent.

In a fifth aspect, the invention provides a method of treating cancer or proliferative disease in a patient comprising administering a substance obtained by screening candidate substances in a method which comprises combining a protein having the amino acid residue sequence shown in Figure 2 (SEQ ID NO.: 2) and Cdc25 protein under phosphorylating conditions, contacting the resulting o008 COMS ID No: SBMI-06388059 Received by IP Australia: Time 14:29 Date 2007-02-26 26/02 '07 MON 14:17 FAX 61299255911 GRIFFITH HACK 0oo9 o 2

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0 combination with a candidate substance, and determining any change in Sprotein phosphorylation activity in order to identify candidate substances which modulate phosphorylation activity, and selecting a candidate substance which modulates phosphorylation activity to thereby obtain the substance, to a patient in need thereof.

n In one embodiment, the method further comprises administering a DNA Cn damaging chemotherapeutic agent.

o In one embodiment, the substance is administered by administering a pharmaceutical composition comprising the substance together with a pharmaceutically acceptable carrier, diluent or excipient therefor. The composition may further comprise a DNA damaging chemotherapeutic agent.

In a sixth aspect, the invention provides use of a compound that is an inhibitor or activator of expression of the CDSI human cell cycle checkpoint pathway protein comprising the amino acid sequence depicted in Figure 2 (SEQ ID NO: in the manufacture of a medicament for treating cancer or proliferative disease, wherein the compound is identified by a method which comprises contacting a cell expressing the proteins of the CDS1 human cell cycle checkpoint pathway with said compound, and comparing the level of expression of the CDS1 human cell cycle checkpoint pathway protein of said cell against a 2 o cell which has not been in contact with said compound, wherein said compound increases or decreases expression of the CDS 1 human cell cycle checkpoint pathway protein.

In a seventh aspect, the invention provides use of a compound that is an inhibitor or activator of expression of the CDS1 human cell cycle checkpoint pathway protein comprising the amino acid sequence depicted in Figure 2 (SEQ ID NO: and a DNA damaging chemotherapeutic agent, in the manufacture of a medicament for treating cancer or proliferative disease, wherein the compound is identified by a method which comprises contacting a cell expressing the proteins of the CDS1 human cell cycle checkpoint pathway with said compound, COMS ID No: SBMI-06388059 Received by IP Australia: Time 14:29 Date 2007-02-26 26/02 '07 MON 14:17 FAX 61299255911 GRIFFITH HACK [Oio o 2E and comparing the level of expression of the CDS1 human cell cycle checkpoint f, pathway protein of said cell against a cell which has not been in contact with said N compound, wherein said compound increases or decreases expression of the CDS 1 human cell cycle checkpoint pathway protein.

or In an eighth aspect, the invention provides use of a substance obtained en by screening candidate substances in a method which comprises: eCn providing a protein comprising the amino acid sequence of SEQ ID NO.: 2 or Sa functional equivalent, wherein said functional equivalent comprises conservative substitutions, deletions or insertions of the amino acid sequence of Figure 2 such that said protein comprises different amino acids than those of the amino acid sequence shown in Figure 2, yet which substitutions, deletions or insertions do not affect the activity of the protein having the sequence of Figure 2 (SEQ ID NO: as measured by the ability to interact in the human cell cycle checkpoint pathway, wherein the protein or equivalent of said protein exhibits a kinase activity; contacting a substrate for said protein with the protein under conditions such that the kinase will act upon the substrate; bringing the protein and substrate into contact with a candidate substance; measuring the degree of increase or decrease in the kinase activity of the protein; and selecting a candidate substance which provides a decrease or increase in said kinase activity to thereby obtain the substance, in the manufacture of a medicament for treating cancer or proliferative disease.

In a ninth aspect, the invention provides use of a substance obtained by screening candidate substances in a method which comprises: providing a protein comprising the amino acid sequence of SEQ ID NO.: 2 or a functional equivalent, wherein said functional equivalent comprises conservative substitutions, deletions or insertions of the amino acid sequence of Figure 2 such that said protein comprises different amino acids than those of the COMS ID No: SBMI-06388059 Received by IP Australia: Time 14:29 Date 2007-02-26 26/02 '07 MON 14:18 FAX 61299255911 GRIFFITH HACK 2F

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amino acid sequence shown in Figure 2, yet which substitutions, deletions or V,0 insertions do not affect the activity of the protein having the sequence of Figure 2 (SEQ ID NO: as measured by the ability to interact in the hunan cell cycle checkpoint pathway, wherein the protein or equivalent of said protein exhibits a n 5 kinase activity; n contacting a substrate for said protein with the protein under conditions such c N that the kinase will act upon the substrate; S(c) bringing the protein and substrate into contact with a candidate substance; c measuring the degree of increase or decrease in the kinase activity of the protein; and selecting a candidate substance which provides a decrease or increase in said kinase activity to thereby obtain the substance, and a DNA damaging chemotherapeutic agent, in the manufacture of a medicament for treating cancer or proliferative disease.

In a tenth aspect, the invention provides use of a substance obtained by screening candidate substances in a method which comprises: providing a protein comprising the amino acid sequence of SEQ ID NO.: 2 or a functional equivalent, wherein said functional equivalent comprises conservative substitutions, deletions or insertions of the amino acid sequence of Figure 2 such that said protein comprises different amino acids than those of the amino acid sequence shown in Figure 2, yet which substitutions, deletions or insertions do not affect the activity of the protein having the sequence of Figure 2 (SEQ ID NO: as measured by the ability to interact in the human cell cycle checkpoint pathway, wherein the protein or equivalent of said protein exhibits a kinase activity; contacting a substrate for said protein with the protein under conditions such that the kinase will act upon the substrate; bringing the protein and substrate into contact with a candidate substance; ioil COMS ID No: SBMI-06388059 Received by IP Australia: Time 14:29 Date 2007-02-26 26/02 '07 MON 14:18 FAX 61299255911 GRIFFITH HACK [012 t27-- S(d) measuring the degree of increase or decrease in the kinase activity of the INO protein; and C selecting a candidate substance which provides a decrease or increase in said kinase activity to thereby obtain the substance, Sin the manufacture of a medicament for increasing susceptibility of cancer cells tr n to chemotherapy and/or radiotherapy.

c, In an eleventh aspect, the invention provides use of a compound capable 0 of modifying the activity of CDS 1 human cell cycle checkpoint pathway protein comprising the amino acid sequence depicted in Figure 2 (SEQ ID NO: in the manufacture of a medicament for treating cancer or proliferative disease in a patient, wherein the compound is identified by a method which comprises combining Cdc25 protein with human hCDS1 protein in the presence of ATP and the said compound, and detecting Cdc25 phosphatase activity to thereby obtain said compound.

is In a twelfth aspect, the invention provides use of a compound capable of modifying the activity of CDS human cell cycle checkpoint pathway protein comprising the amino acid sequence depicted in Figure 2 (SEQ ID NO: and a DNA damaging chemotherapeutic agent, in the manufacture of a medicament for treating cancer or proliferative disease in a patient, wherein the compound is identified by a method which comprises combining Cdc25 protein with human hCDS 1 protein in the presence of ATP and the said compound, and detecting phosphatase activity to thereby obtain said compound.

In a thirteenth aspect, the invention provides use of a substance obtained by screening candidate substances in a method which comprises combining a protein having the amino acid residue sequence shown in Figure 2 (SEQ ID NO.: 2) and Cdc25 protein under phosphorylating conditions, contacting the resulting combination with a candidate substance, and determining any change in protein phosphorylation activity in order to identify candidate substances which modulate phosphorylation activity, and selecting a candidate substance which COMS ID No: SBMI-06388059 Received by IP Australia: Time 14:29 Date 2007-02-26 26/02 '07 MON 14:19 FAX 61299255911 GRIFFITH HACK 2 1013 2H modulates phosphorylation activity to thereby obtain the substance, in the manufacture of a medicament for treating cancer or proliferative disease in a patient.

In a fourteenth aspect, the invention provides use of a substance obtained s by screening candidate substances in a method which comprises combining a protein having the amino acid residue sequence shown in Figure 2 (SEQ ID NO.: 2) and Cdc25 protein under phosphorylating conditions, contacting the resulting COMS ID No: SBMI-06388059 Received by IP Australia: Time 14:29 Date 2007-02-26 26/02 '07 MON 14:19 FAX 61299255911 GRIFFITH HACK Z014 0 combination with a candidate substance, and determining any change in protein phosphorylation activity in order to identify candidate substances N which modulate phosphorylation activity, and selecting a candidate substance which modulates phosphorylation activity to thereby obtain the substance, and a n DNA damaging chemotherapeutic agent, in the manufacture of a medicament for Streating cancer or proliferative disease in a patient.

Described herein is a compound that is an inhibitor or activator of Sexpression of the CDS I human cell cycle checkpoint pathway protein comprising the amino acid sequence depicted in Figure 2 (SEQ ID NO: when identified by a method which comprises contacting a cell expressing the proteins of the CDS I human cell cycle checkpoint pathway with said compound, and comparing the level of expression of the CDS 1 human cell cycle checkpoint pathway protein of said cell against a cell which has not been in contact with said compound, wherein said compound increases or decreases expression of the CDS 1 human cell cycle checkpoint pathway protein.

Also described herein is a substance for use in anti-cancer therapy when obtained by screening candidate substances in a method which comprises: providing a protein comprising the amino acid sequence of SEQ

ID

NO: 2 or a functional equivalent, wherein said functional equivalent comprises conservative substitutions, deletions or insertions of the amino acid sequence of Figure 2 such that said protein comprises different amino acids than those of the amino acid sequence shown in Figure 2, yet which substitutions, deletions or insertions do not affect the activity of the protein having the sequence of Figure 2 (SEQ ID NO: as measured by the ability to interact in the human cell cycle checkpoint pathway, wherein the protein or equivalent of said protein exhibits a kinase activity; contacting a substrate for said protein with the protein under conditions such that the kinase will act upon the substrate; COMS ID No: SBMI-06388059 Received by IP Australia: Time 14:29 Date 2007-02-26 26/02 '07 MON 14:19 FAX 61299255911 GRIFFITH HACK Q 015 4 bringing the protein and substrate into ontact with a candidate substance; decrease in the kinase activity of measuring the degree of increase or decreasof the prot selecting a candidate substance which provides a decrease or increase in said kinase activity to thereby obtain the substance.

Also described herein is a compound capable of modifying.the activity of CDS 1 human cell cycle checkpoint pathway protein comprisig the amino acid sequence depicted in Figure 2 (SEQ ID NO: 2) when identified by a method o1 which comprises combining Cdc25 protein with human hCDS1 protein in the presence of ATP and the said compound, and detecting Cdc25 phosphatase activity to thereby obtain said compound.

Also described herein is a substance suitable for anti-cancer therapy, b ei a muethod which compnses when obtained by screening candidate substances in a method which comprises comlbining a protein having the amino acid residue sequence shown in Figure 2 (SEQ ID NO: 2) and Cdc25 protein under phosphorylating conditions, cotacting the resulting combination with a candidate substance, and determining any change in Cdc25 protein phosphorylation activity in order to identify candidate substances which modulate phosphorylation activity, and selecting a candidate 0 substance which modulates phosphorylation activity to thereby obtain the substance.

Described herein is the discovery of a novel human checkpoint kinase gene hCDS 1, protein and constructs and methods for the production and use of hCDS CDS Described herein is a nucleic acid sequence which encodes for hCDS1, consisting of the nucleic acid sequence of SEQ ID NO: In particular, described herein is the nucleic acid sequence from position 66 to 1694 of the nucleic acid sequence of SEQ ID NO:1, which translates into the hCDS1 protein.

Also described herein are nucleic acid constructs, vectors, plasmids, cosmids and COMS ID No: SBMI-06388059 Received by IP Australia: Time 14:29 Date 2007-02-26 26/02 '07 MON 14:19 FAX 61299255911 GRIFFITh HACK 018 26/02 '07 MON 14:19 FAX 61299255911 GRIFFITH HACK o1016 0 0 the like which contain the nucleic acid sequence of SEQ ID NO: In particular, described herein are nucleic acid vector constructs which contain the nucleic aci sequence of SEQ ID NO:1 and are capable of expressing protein from this eq s 1- acid vectors that are nucleic acid sequence. Also described herein are nucleic acid vectors that are n 5 suitable for the transformation of host cells, whether eukaryotic or prokaryotic, suitable for incororat into viral vectors, or suitable for in vitro protein suitable for incorporation intof SEQ D NO:1 in n ,expression. Also described herein is nucleic acid sequence of SEQ NO:1 in Standem with, or otherwise in conjunction with additional nucleic acids for the generation of fusion protein products containing at least the functional segment lo of the protein encoded for by the nucleic acid of SEQ ID NO: 1. Also described herein is the nucleic acid of SEQ ID NO:I adapted for use as a naked DNA transformant for incorporation and expression in target cells. Also described herein are anti-sense DNA molecule formulations which are the complement to the nucleic acid sequence of SEQ ID NO: 1, and fragments thereof, whether complementary to contiguous or discontinuous portions of the nucleic acid sequence of SEQ ID NO: Also described herein are compositions incorporating modified nucleotides or backbone components which encode for the nucleic acid sequence of SEQ ID NO: 1, its complement, or fragments thereof. Such modified nucleotides and nucleic acids are known in the art (see for example Verma et al., Ann. Rev. Biochen. 67: 99-134 (1998)). Thus described herein are modified nucleic acids which incorporate, for example, internucleotide linkage modification, base modifications, sugar modification, non-radioactive labels, nucleic acid cross-linking, and altered backbones including PNAs (polypeptide nucleic acids).

Also described herein is the novel human checkpoint kinase protein hCDS1, which consists of the amino acid sequence of SEQ ID 140:2. Also described herein is hCDS1 protein produced by recombinant DNA technology and expressed in vivo or in vitro. Also described herein is hCDS1 protein produced by transformed host cells in small-scale or large-scale production.

produced b. ^an COMS ID No: SBMI-06388059 Received by IP Australia: Time 14:29 Date 2007-02-26 I~J 017 26/02 '07 MON 14:20 FAX 61299255911 GRIFFITH HACK S6 Also described herein is hCDS 1 protein, in either glycosylated or unglycosylated ID forms, produced by either eukaryotic or prokryotic cell Also described herein Sis hCDS 1 protein expressed from mammalian, insect, plant, bacterial, fungal, or kn any other suitable host cell. Also described herein is hCDS I protein that is Sproduced as a fusion protein product, conjugated to a solid support, or hCDS1 n protein which is labeled with any chemical, radioactive, fluorescent, n. chemiluminescent or otherwise detectable marker. Also described herein is o hCDS1 protein isolated from natural sources and enriched in purity over that found in nature. Also described herein are pharmaceutical formulations of So hCDS1 protein and formulations of the hCDS1 protein pharmaceutically acceptable carriers or excipients.

Also described herein is a nucleic acid sequence which would encode for the amino acid sequence of SEQ ID NO:2, and the embodiments of these nucleic acid sequences as described for SEQ ID NO:1, as the nucleic acid code for is generating any nucleic acid sequence which will encode for a protein having the amino acid sequence of SEQ ID NO:2 is predictable to one of skill in the art.

Also described herein are antibodies which bind specifically to the hCDS 1 protein, either polyclonal or monoclonal, as generated by the immunization of a mammal with protein having the amino acid sequence of SEQ ID NO:2, or fragments thereof.

Also described herein are equivalent proteins where substitutions of amino acids in the sequence of SEQ ID NO:2 that are reasonably predictable as being equivalent, and the embodiments thereof as described for SEQ ID NO:2.

For example, non-polar (hydrophobic side-chain) amino acids alanine, valine, leucine, isoleucine, proline, phenylalanine, tryptophan, methionine; uncharged polar amino acids glycine, serine, threonine, cysteine, tyrosine, asparagne, glutamine; charged polar amino acids aspartic acid, glutamic acid; basic amino acids lysine, arginine, and histidine are understood by those in the art to have functionally predictable effects when substituted. Thus also described herein are [017 COMS ID No: SBMI-06388059 Received by IP Australia: Time 14:29 Date 2007-02-26 26/02 '07 MON 14:20 FAX 61299255911 GRIFFITh HACK ~JO18 26/02 '07 MON 14:20 FAX 61299255911 GRIFFITH HACK 0o018 7 equivalent nucleic acids which encode for such equivalent proteins and the Sembodilients thereof as described for SEQ ID NO:1.

Also described herein are methods of generating hCDS I protein, by using recombinant DNA technology and the appropriate nucleic acid encoding tr ents therof. Described herein is for hCDS1 protein, fusion protein, or fragments thereof Described herein is incorporating an appropriate nucleic acid sequence into a suitable expression en vector, together with the incorporation of any suitable control elements such as a v r tgether with the incorpration Also Spromoter, and/or an enhancer, either inducible or constitutively expressed. Also Sdescribed herein is the use of expression vectors with or without at least one additional selectable marker or expressible protein. Also described herein are methods wherein a suitably constructed expression vector is transformed or oerwise introduced into a suitable host cell, and protein is expressed by such a host cell. Thus, described herein are transformed host cells, which are capable of producing hCDS protein, fusion protein, or fragments thereof.

1s The discovery that hCDS 1 acts in coordination with Cdc25 in the DNA damage checkpoint allows for the use of compounds in methods for therapeutic treatment of diseases which involve abnormal DNA damage checkpoint function.

Described herein is the use of the compounds as therapeutics for the treatment of cancer. In particular, described herein is the specific modification of the hCDS 1- Cdc25 DNA damage checkpoint in cells.

Also described herein are methods for screening test compounds for efficacy in effecting the hCDS mediated checkpoint function of eukaryotic cells, said method comprising contacting a test compound to eukaryotic cells, and detecting any change in hCDS1 expression or function. Thus, described herein is a method of screening wherein said detection of change in hCDS 1 expression or function is accomplished by assaying for hCDS mRNA production, or by assaying for hCDSI protein expression. In particular, described herein is the screening of candidate substances for efficacy in modifying the DNA damage checkpoint by screening for any change in COMS ID No: SBMI-06388059 Received by IP Australia: Time 14:29 Date 2007-02-26 26/02 '07 MON 14:20 FAX 61299255911 GRIFFITH HACK i019 S8 0 phosphorylation, or kinase activity. The compounds or substances identified by the assays, or compounds corresponding to such compounds or substances, can be used for the manufacture of pharmaceutical therapeutics.

Thus, described herein are pharmaceutical compositions which include o 5 the hCDSI protein, hCDS1 nucleic acid, hCDSI anti-sense nucleic acids. Also ci described herein are compounds or substances identified as suitable for use as a 0 therapeutic by the assays, in pharmaceutical formulations. These pharmaceutical Scompositions can further include chemotherapeutic agents for the use in treating cancer, or be administered in a regimen coordinated with the administration of other anti-cancer therapies. Described herein are methods for combined chemotherapy using the hCDS1 derived pharmaceuticals independently, and in combination with other chemotherapeutic agents, and in a second embodiment as admixtures with other anti-cancer therapeutics for single dose administration.

Described herein is a nucleic acid encoding hCDS1 protein having the amino acid sequence illustrated in Figure 2 (SEQ ID NO:2), or encoding a functional equivalent fragment, or bioprccursor of said protein. The nucleic acid may be a DNA molecule such as a genomic DNA molecule or, may be a cDNA molecule, however it may also be RNA.

A nucleic acid encoding hCDS 1 protein may comprise the nucleic acid sequence represented by position 66 to 1694 of the sequence illustrated in Figure 1 (SEQ ID NO:1), the complement thereof, or a nucleic acid sequence capable of hybridizing to either under high stringency conditions.

The nucleic acid sequences described herein may be capable of hybridizing under low stringency conditions to nucleic acid sequences derived from family members to identify homologues therefrom or alternatively to identify nucleic acid sequences from other species.

As would be well known to those skilled in the art due to the degeneracy of the genetic code the nucleic acid sequences described herein may include substitutions therein yet which still encode the same amino acid sequence.

COMS ID No: SBMI-06388059 Received by IP Australia: Time 14:29 Date 2007-02-26 26/02 '07 MON 14:21 FAX 61299255911 GRIFFITH HACK 1020 O9

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The nucleic acids described herein may be incorporated into an Sexpression vector and may be subsequently used to transform, transfect or infect a suitable host cell. In such an expression vector the nucleic acid described herein may be operably linked to a control sequence, such as a suitable promoter in ex n s lerot cell. The vector are also described herein. The host cell may be a eukaryotic cell or a c bacterial cell, and may even be a mammalian cell or insect cell. Mammalian host cells are particularly advantageous because they provide the necessary posttranslational modifications to the expressed proteins, such as glycosylation or the like, which modifications confer optimal biological activity on said proteins, which when isolated may advantageously be used in diagnostic kits or the like.

The expression vector including said nucleic acid may be used in vivo, such as in, for example, gene therapy.

As described herein there is also a transgenic cell, tissue or organism comprising a transgene capable of expressing hCDS 1 protein, which protein comprises the amino acid sequence illustrated in Figure 2 (SEQ ID NO:2), or the COMS ID No: SBMI-06388059 Received by IP Australia: Time 14:29 Date 2007-02-26 9A amino acid sequence of a functional equivalent or bioprecursor or fragment therefor. The term "transgene capable of expression" as used herein means a suitable nucleic acid sequence which leads to expression of hCDS 1 or proteins, having the same function and/or activity. The transgene may include, for example, genomic nucleic acid isolated from human cells or synthetic nucleic acid, including DNA integrated into the genome or in an extrachromosomal state.

The transgene may comprise the nucleic acid sequence encoding the proteins described herein, or a functional fragment of said nucleic acid. A functional fragment of said nucleic acid should be taken to mean a fragment of the gene comprising said nucleic acid coding for the proteins described herein or a functional equivalent, derivative or a non-functional derivative such as a dominant negative mutant, or bioprecursor of said proteins. For example, it would be readily apparent to persons skilled in the art that nucleotide substitutions or deletions may be used using routine techniques, which do not affect the protein sequence encoded by said nucleic acid, or which encode a functional protein according to the invention.

The hCDS 1 protein expressed by said transgenic cell, tissue or organism or a functional equivalent or bioprecursor of said protein is also described herein.

Also described herein is an antisense molecule which is capable of hybridizing to the nucleic acid described herein. Advantageously, the antisense molecule may be used as a medicament, or in the preparation of a medicament for the treatment of cancer and other proliferative diseases.

Also described herein are nucleic acid sequences of at least approximately 15 nucleotides of a nucleic acid described herein, and may be from 15 to 50 nucleotides. These sequences may be used as probes or primers to initiate replication, or the like. Such nucleic acid sequences may be produced according to techniques well known in the art, such as by recombinant or synthetic means. They may also be used in diagnostic kits or the like for detecting the presence of a nucleic acid. These tests generally comprise contacting the probe with the sample under hybridizing conditions and detecting 9B for the presence of any duplex or triplex formation between the probe and any nucleic acid in the sample.

The nucleic acid sequences described herein may be produced using such recombinant or synthetic means, such as for example using PCR cloning mechanisms which generally involve making a pair of primers, which may be from approximately 15 to 50 nucleotides spanning a region of the gene which is desired to be cloned, bringing the primers into contact with mRNA, cDNA, or genomic DNA from a human cell, performing a polymerase chain reaction under conditions which bring about amplification of the desired region (and where necessary first performing a reverse transcription step), isolating the amplified region or fragment and recovering the amplified DNA. Generally, such techniques as defined herein are well known in the art, such as described in Sambrook et. al., (Molecular Cloning; a Laboratory Manual, 1989). Human allelic variants of the nucleic acid described herein may be obtained by for example, probing genomic DNA libraries from a range of individuals for example from different populations, and other genotyping techniques.

Furthermore, nucleic acids and probes may be used to sequence genomic DNA from patients, using techniques well known in the art, for example, the Sanger dideoxy chain termination method, which may ascertain any predisposition of a patient to certain proliferative disorders.

Also described herein are isolated proteins having the amino acid sequences as illustrated in Figure 2 (SEQ ID NO:2) or the amino acid sequence of a functional equivalent functional fragment or bioprecursor of said protein in addition to isolated antibodies, monoclonal or polyclonal capable of binding to the amino acid sequences of these proteins or fragments thereof. As would be well known to those skilled in the art, the proteins described herein may comprise conservative substitutions, deletions or insertions wherein the protein comprises different amino acids than those disclosed in Figure 2, yet which 9C substitutions, deletions or insertions do not affect the activity of the proteins or their ability to interact in the human cell cycle checkpoint pathway.

Fragments may include those comprising an epitope of the proteins described herein. The epitopes may be determined using, for example, peptide scanning techniques as described in Geysen et. al., Mol. Immunol., 23; 709-715 (1986).

The antibodies described herein may be produced according to techniques which are known to those skilled in the art. Monoclonal antibodies may be prepared using conventional hybridoma technology as described in Kohler F and Milstein C (1985), Nature 256, 495-497. Polyclonal antibodies may also be prepared using conventional technology well known to those skilled in the art, and which comprises inoculating a host animal, such as a mouse, with a protein or epitope described herein and recovering the immune serum. Also described herein are fragments of whole antibodies which maintain their binding activity, such as for example, Fv, F(ab') and F(ab')2 fragments as well as single chain antibodies.

The nucleic acid and/or the proteins described herein may be included in a pharmaceutical composition together with a pharmaceutically acceptable carrier, diluent or excipient therefor. The pharmaceutical composition containing said nucleic acids may, for example, be used in gene therapy. Such nucleic acids, described herein, may be administered naked, or packaged in protein capsules, lipid capsules, liposomes, membrane based capsules, virus protein, whole virus, cell vectors, bacterial cell hosts, altered mammalian cell hosts, or other such suitable means for administration.

Also described herein is a method for detecting for the presence or absence of a nucleic acid described herein, in a biological sample, which method comprises, a) bringing said sample into contact with a probe comprising a nucleic acid or probe described herein under hybridizing conditions, and b) detecting for the presence of hybridization, for example, by the presence of any duplex or triplex formation between said probe and any nucleic acid present in 10 said sample. Proteins described herein may also be detected by a) contacting said sample with an antibody to an epitope of a protein as described herein under conditions which allow for the formation of an antibody-antigen complex, b) monitoring for the presence of any antigen-antibody complex.

Kits for detecting said nucleic acids and proteins are also described herein. A kit for detecting for the presence of a nucleic acid as described herein in a biological sample may comprise means for contacting the sample with a probe comprising a nucleic acid or a probe as described herein and means for detecting for the presence of any duplex or triplex formation between said probe and any nucleic acid present in the sample.

Likewise, a kit for detecting for the presence of a protein as described herein in a biological sample may comprise means for contacting said sample with an antibody to an epitope of a protein as described herein under conditions which allow for the formation of an antibody protein complex, and means for monitoring said sample for the presence of any protein antibody complex.

Also described herein is a method of determining whether a compound is an inhibitor or an activator of expression or activity of the proteins of the human cell cycle checkpoint pathway which method comprises contacting a cell expressing the proteins in said pathway with said compound and comparing the level of expression of any of the proteins of the checkpoint pathway of said cell against a cell which has not been contacted with said compound. Any compounds identified may then advantageously be used as a medicament or in the preparation of a medicament for treating cancer or proliferative disorders.

Alternatively, the compounds may be included in a pharmaceutical composition together with a pharmaceutically acceptable carrier, diluent or excipient therefore. Advantageously, any compounds identified as an inhibitor of the cell checkpoint pathway may be included in a pharmaceutical composition together with a cytotoxic agent, such as a DNA damaging chemotherapeutic agent, and a pharmaceutically acceptable diluent or excipient therefore. Thus, the human cell 11 cycle checkpoint inhibitor may enhance the chemotherapeutic effect of cytotoxic agents used in, for example, anti-cancer therapy.

Also described herein is a method for screening candidate substances for anti-cancer therapy, which method comprises a) providing a protein as described herein exhibiting kinase activity together with a substrate for said protein under conditions such that the kinase will act upon the substrate, b) bringing the protein and substrate into contact with a candidate substance, c) measuring the degree of any increase or decrease in the kinase activity of the protein, and d) selecting a candidate substance which provides a decrease or increase in activity. Such a candidate substance may also be used as a medicament, or in the preparation of a medicament for the treatment of cancer or other such proliferative cell disorders.

Also described herein is a method of identifying other proteins active in the cell checkpoint pathway, which method comprises a) contacting a cell extract with an antibody to an epitope of a protein as described herein, under appropriate binding conditions, b) identifying any antibody-protein complex and c) analyzing the complex to identify any protein bound to the antibody or protein which is other than the protein described herein.

Another method for identifying proteins involved in the cell checkpoint pathway utilizes a two-hybrid system developed in yeast by Chien et. al., supra (1991). This technique is based on functional in vivo reconstitution of a transcription factor which activates a reporter gene. More particularly the technique comprises providing an appropriate host cell with a DNA construct comprising a reporter gene under the control of a promoter regulated by a transcription factor having a DNA binding domain and an activating domain, expressing in the host cell a first hybrid DNA sequence encoding a first fusion of a fragment or all of a nucleic acid sequence and either said DNA binding domain or the activating domain of the transcription factor, expressing in the host cell at least one second hybrid DNA sequence encoding putative binding proteins to be investigated together with the DNA binding domain or activating domain of the transcription factor which is not incorporated in the first fusion; detecting any 12 binding of the protein being investigated with a protein described herein by detecting for the production of any reporter gene product in the host cell; optionally isolating second hybrid DNA sequences encoding the binding protein.

The method may comprise: constructing at least two nucleotide vectors, the first of which comprises a nucleotide segment encoding for a DNA binding domain of GAL4 protein operably linked to a nucleic acid sequence encoding a protein as described herein, the second vector comprising a nucleotide sequence encoding a protein binding domain of GAL4 operably linked to a nucleotide sequence encoding a protein to be tested, co-transforming each of said vectors into a yeast cell being deficient for transcription of genes encoding galactose metabolizing proteins, wherein interaction between said test protein and the protein described herein leads to transcription of galactose metabolic genes.

BRIEF DESCRIPTION OF THE DRAWINGS The following examples are given by way of example only, with reference to the accompanying drawings, wherein: Figure 1 illustrates the nucleotide sequence of hCDS 1 cDNA (SEQ ID NO:l) wherein residues 66-1694 are the coding regions, and depicts the 3' and untranslated regions (UTRs). The initiation and termination codons are shown in bold.

Figure 2 illustrates the deduced amino acid sequence of hCDS 1 (SEQ ID NO:2).

Figure 3 illustrates the amino acid sequence alignment of hCDS 1 and S.

pombe cds 1 performed using the CLUSTALW alignment program and annotated using the GENEDOC program. Residues shaded black are identical between the two proteins and residues shaded grey are similar.

12A DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Described herein is the isolation and characterization of a novel human checkpoint kinase gene and protein which is called hCDS 1. The hCDS gene and protein show some similarity to a homologous gene and protein found in S. pombe.

The S. pombe cdsl+ gene was identified by its ability to complement a DNA polymerase a mutant (Murakami Okayama, 1995, Nature, 374: 817- 819). S. pombe cdsl was also able to suppress the hydroxyurea sensitivity (DNA replication-dependent checkpoint) of rad 1, rad3 and rad9 mutant S. pombe strains but not the UV sensitivity (DNA damage-dependent checkpoint). This shows that S. pombe cds executes its checkpoint function during DNA synthesis.

S. pombe cdsl is a putative protein kinase that is 70% similar to the S.

cerevisiac checkpoint gene RAD53. In S. cerevisiae the DNA damage- and DNA replication-dependent checkpoints are genetically separate at the level of detection of DNA lesions. The two pathways then converge on the Rad53 protein kinase which potentially acts as an amplifier in the signal transduction pathway. This appears not to be the case in S. pombe where the same proteins are involved in detection of all types of lesion but the transduction of the signal follows separate pathways involving different protein kinases; S. pombe cdsl for the DNA replication-dependent checkpoint and Chkl/Rad27 for the DNA damage-dependent pathway. It has been suggested that S-phase-specific activation of cdsl kinase may define a subpathway of the checkpoint response in S. pombe (Lindsay et al., 1998, Genes and Development, 12: 382-395).

S. pombe cdsl may act via an interaction with DNA polymerase a to monitor the progress of DNA replication or the integrity of replication complexes. There is evidence in Drosophila for a kinase of the appropriate molecular weight associating with DNA polymerase a (Peck et al., 1993, 190: 325-331). Alternatively it may act via phosphorylation of pl07' 1 in a manner analogous to Chkl ultimately affecting the activity of the Gl/S phase cyclin dependent kinases.

Many of the methods and materials for carrying out the basic molecular biology manipulations as described in the examples below are known in the art, and can be found in such references as Sambrook et al., Molecular Cloning, 2nd edition, Cold Spring Harbor Laboratory Press (1989); Berger et al., Guide to Molecular Cloning Techniques, Methods in Enzymology, Vol. 152, Academic Press, Inc., (1987); Davis et al., Basic Methods in Molecular Biology, Elsevier Science.Publishing Co., Inc. (1986); Ausubel et al., Short Protocols in Molecular Biology, 2nd ed., John Wiley Sons, (1992); Goeddel Gene Expression Technoloyv, Methods in Enzymology, Vol.

185, Academic Press, Inc., (1991); Guthrie et al., Guide to Yeast Genetics and Molecular Biolowy. Methods in Enzymology, Vol. 194, Academic Press, Inc., (1991); McPherson et al., PCR Volume 1, Oxford University Press, (1991).

The invention in its several aspects can be more readily understood by reviewing the following examples.

Example 1 Isolation of hCDS1 Isolation of hCDS1 began with a search for sequences similar to S. pombe cdsl+ using the TBLASTN program. A human expressed sequence tag (EST No. 864164) was identified in the proprietary LifeSeq(R) database (Incyte Pharmaceuticals Inc., Palo Alto, CA, USA). Sequence analysis of the 1.3 kb insert revealed an incomplete open reading frame which was similar to S. pombe cdsl.

Approximately 650 nucleotides of novel 5' DNA sequence was obtained by 5'RACE (rapid amplification of cDNA ends) using a Marathon Ready human placental cDNA (Clontech), following the manufacturer's instructions.

Briefly, the two hCDS1 gene specific primers used for nested PCR (Polymerase chain reaction) reactions were GSP3 5'-TTTTGCTGATGATCTTTATGGCTAC-3' (SEQ ID NO.: 3) and GSP4 CACAGGCACCACTTCCAAGAGTTTT-3' (SEQ ID NO.: 4).

Subsequently, a complete ORF for hCDS1 was amplified from a human SK-N-MC neuroblastoma cDNA library using the PCR primers 5'-GGGCTCGAGAGCAGCGATGTCTCGGGAGTCGGATGT-3' (SEQ ID NO.: 5) and 5'-GGCGGATCCTCGAGTCACAACACAGCAGCACACAC-3'

(SEQ

ID NO.: The amplification product was then cloned into pCR2.1 vector (Invitrogen) and the DNA sequence determined.

The nucleic acid sequence of hCDS1 was found to show 47.8% identity to the S. pombe cdsl+ at the DNA level.

Termination codons were present in all three reading frames in the 120 nucleotides immediately 5' to the putative hCDSl initiation codon, indicating that the complete coding region has been isolated. Parts of the sequence were also found to match partial sequences found in the NCBI databases, EST AA285249, genomic sequence H55451, and the 54 base pair fragment H55698.

The identified human gene and vectors encoding the hCDS1 nucleic acid sequence were deposited as plasmid 14A HCDS1 ORF/pCR-Blunt deposited under Accession No. LMBP 3708; plasmid HCDS1 5'RACE fragment/pGEM-Easy deposited under Accession No. LMBP 3710; and plasmid HCDS1 3'fragment Incyte clone 864164/pSPORT deposited under Accession No. LMBP 3709 with the Belgian Co-ordinated Collections of Micro-organisms (BCCM) at Laboratorium Voor Moleculaire Biologies-Plasmidencollecte (LMBP) 35, B-9000 Gent, Belgium, in iaccordance with the provisions of the Budapest Treaty, 28 April 1997.

The tissue expression profile of hCDS 1 was examined on multiple tissue Northern blots (Clontech) and a cancer cell line Northern blot (Clontech), which were probed with the hCDS ORF. A single transcript of approximately 2.1 kb was observed. Expression was undetectable by conventional Northern blot hybridization conditions in all normal human tissues examined. However, expression was found to be greatly elevated in all of the cancer cell lines examined.

The hCDS1 gene was localized to chromosome 22q 1.2-q12, as determined using the complete ORF as a probe for FISH (Fluorescent in situ Hybridization) analysis. The hybridization efficiency was approximately 62%, and no other loci were detected under the conditions used.

Briefly, lymphocytes isolated from human blood were cultured in c-minimal essential media (MEM) supplemented with 10% fetal calf serum and phytohaemagglutinin (PHA) at 37oC for 68-72 hours. The lymphocyte cultures were treated with BrdU (0.18 mg/ml, Sigma) to synchronize the cell population. The synchronized cells were washed three times with serum-free medium to release the block and re-cultured at 37°C for 6 hours in a-MEM with thymidine (2.5 1ig/ml Sigma). Cells were harvested and slides were prepared using standard procedures including hypotonic treatment, fixation and air-drying. DNA fragments containing the hCDSI complete ORF were gel purified and biotinylated with dATP using the BRL BioNick labeling kit (15-C, 1 hour) (Heng et al., 1992, Proc. Natl. Acad. Sci. USA.

89: 9509-9513).

Slides were then baked at 55-C for 1 hour, and after RNase treatment, the slides were denatured in 70% formamide in 2x SSC for 2 minutes at 70C followed by dehydration with ethanol. Probes were denatured at 75-C for 5 minutes in a hybridization mix consisting of 50% formamide and 10% dextran sulphate. Probes were loaded on the denatured chromosomal slides. After overnight hybridization, slides were washed and detected. FISH signals and the DAPI-banding pattern were 3 0 recorded separately by taking photographs, and the assignment of the FISH mapping data with chromosomal bands was achieved by superimposing FISH signals with DAPI banded chromosomes (Heng Tsui, 1994, Methods in Mol. Biol.. 33: 35-49).

Example 2 Characterization of hCDS1 protein The hCDS1 nucleic acid sequence cDNA predicts a translation product of 543 amino acids with an approximate molecular weight of 61kDa. This is close to the apparent molecular weight of endogenous Cdsl protein in HeLa cells.

The predicted hCDS1 protein, is 28% identical to the cdsl protein of S. pombe, 28% identical to RAD53 and 27% identical to the DUN1 kinase of S. cerevisiae. Sequence alignment of these apparent homologues shows several regions of sequence similarity outside the kinase domain, including conservation of the Fork Head Associated domain (Hoffmann et al., 1995, Trends Biochem. Sci., 20: 347-9).

The human protein shows the same overall structure as S.

pombe CDS1 and S. cerevisiae DUN1 in that it lacks the long C-terminal extension found in RAD53. Northern blot analysis with hCDS1 identified a single transcript of about 2.2 kb expressed in testis and in 8 human cancer samples examined.

Briefly, two multiple tissue Northern blots (Clontech) and a Cancer Cell line Northern blot (Clontech) were hybridized with a cDNA probe for hCDS1. The probe corresponds to the complete ORF as described above. The blots were washed at high stringency (0.1 x SSC, 0.1% SDS, 2 x 20 min) and exposed using Kodak X-OMAT autoradiography film with intensifying screens at Example 3 Cdc25 total activity assay The possibility that dephosphorylation of Cdc2 is down-regulated in the presence of DNA damage required an assay to allow for the analysis of the total activity of In the presence of EDTA, Cdc2/Cyclin B from asynchronous HeLa cell extracts was found to inactivate spontaneously.

Briefly, cells were lysed in ice-cold lysis buffer mM Tris pH 7.4 containing 2 mM magnesium chloride, 1 mM phenylmethylsulphonyl fluoride, and 5 gg/ml leupeptin, pepstatin and aprotinin). Lysates were cleared by centrifugation at 10,000 xg for 10 minutes and the protein concentration of the supernatants determined using the Lowry assay. 10 mM EDTA was added to the supernatants (100 jg in 60 gL) and the reaction initiated by incubation at C. At assay intervals the activity of Cdc2/Cyclin B was assayed by measuring the histone-H1 kinase activity present in anti-Cyclin B immune-precipitates (Blasina et al., supra.). For immunoblots 400 gg of cell lysate was immune-precipitated using anti-Cyclin B antibody, resolved on an 11% acrylamide-SDS gel. Monoclonal antibody against the PSTAIRE motif of Cdc2 was used to detect the different phospho-forms of Cdc2.

Activation correlates with loss of the inhibitedphosphorylated form of Cdc2, visualized as the slower migrating species on SDS-PAGE gels. Activation was prevented by vanadate, an inhibitor of Cdc25 and other tyrosine phosphatase. Furthermore, immune-depletion with anti-sera greatly reduced activation of Cdc2/Cyclin B. There was no increase in the levels of Cdc2 or Cyclin B protein, phosphorylation by WEE1 and Mytl was blocked by the presence of 10 mM EDTA. Thus, these result demonstrate that the activation of Cdc2 was the result of dephosphorylation. In lysates of asynchronous HeLa cells, the endogenous Cdc25 phosphatase activity is sufficient to dephosphorylate and activate more than of the available Cyclin B/Cdc2 in 30 minutes. Analysis of lysates of HeLa cells in which the DNA had been damaged by exposure to 10 Gy of y-irradiation one hour before harvesting showed a significant reduction in the rate of activation of Cdc2, such that less than 25% of the available Cdc2/Cyclin B was activated during the minutes incubation. The amount of Cdc2/Cyclin B in complex was not significantly altered and it was activated to the same extent as control Cdc2/Cyclin B by addition of exogenous GST-Cdc25. Irradiation with 10 Gy led to more than 3-fold reduction in the rate of Cdc2 dephosphorylation in the 10 time courses examined. If the inactivation of Cdc25 measured above is part of the DNAdamage checkpoint response in human cells, then experimental conditions that over-ride the DNA damage checkpoint might be expected to block the radiationinduced inhibition of Example 4 DNA Damage Checkpoint Effect of hCDS1 DNA damage response in a variety of cells is known to require various related kinases which structurally are related to PI-3 kinases. At least one member of the family, DNA-Protein Kinase, has been shown to be sensitive to wortmannin in vitro (Hawley et al., 1996, Genes and Dev., 10: 2383-8; Hartley et al., 1995, Cell, 82: 849- 856). Thus, the possibility that a wortmannin-sensitive kinase acted upstream of the radiation induced delay in Mphase entry was tested (Price et al., 1996, Cancer Research, 56: 246-250). HeLa cells can be arrested in Mphase by nocodazole, irradiation causes cells to delay in G2 prior to the nocodazole-sensitive M-phase block point.

Thus, by scoring the mitotic index of cells that are cultured in nocodazole, it is possible to determine whether entry into mitosis has been delayed. Control cells cultured in the presence of nocodazole for 14 hours contained 60% mitotic cells, the presence of wortmannin had little effect on this number. However, irradiation 18A reduced the number of cells that reach the nocodazole block point to only 10%. In contrast, irradiation in the presence of wortmannin had only a modest effect. These results demonstrate that wortmannin over-rides the DNA damage G2 checkpoint in HeLa cells.

The effects of wortmannin on the radiation-induced inactivation of Cdc25 was then tested. Wortmannin had little effect on the activation of Cdc2/Cyclin B in extracts prepared from unirradiated cultures, however it did greatly diminish the irradiation-induced decrease in activity.

Radiation-induced G2 checkpoint is also over-ridden in cell-lines derived from patients with the genetic disorder ataxia telangiectasia. Ataxia Telangiectasia mutant cells are defective in both the G1 and G2 checkpoints following exposure to many, but not all, agents that damage DNA (Canman et al., 1994, Cancer Research, 54: 5054-5058). The failure of AT-deficient cells to delay G1 correlates with a failure to up-regulate p53 (Kastan et al., 1992, Cell, 71: 587-589), and with failure to phosphorylate and activate cAbl (Baskaran et al., 1997, Nature, 387: 516-519; Shafman et al., 1997, Nature, 387: 520-523). The molecular basis for the failure to delay G2 is unknown. AT-deficient cells show greatly reduced responses to agents that generate chromosomal breaks, such as ionizing y-rays. Remarkably, AT-deficient cells have near normal responses following the base damage that is generated by irradiation with a UV source (Canman et al., 1994, Cancer Research, 54: 5054- 5058; Painter et al., 1980, Proc. Natl. Acad. Sci. USA, 77: 7315-7317; Zampetti-Bosseler et al., 1981, Int. J.

Radiat. Biol., 39: 547-558). The effects of UV and yirradiation on the Cdc25 activity of AT-plus and AT-minus 18B human fibroblast cell-lines was tested.

AT-minus cells respond to UV-irradiation with a robust reduction in the rate at which Cdc2 is dephosphorylated.

In contrast, y-irradiation had only a modest effect on the rate of dephosphorylation of Cdc2. In AT-plus cells the rate of dephosphorylation of Cdc2 was significantly reduced following either ionizing-radiation or UVradiation. These data indicate that the ATM gene product is required for the efficient inactivation of following y-irradiation and demonstrate a correlation between inactivation of Cdc25 and delayed entry into Mphase following DNA damage.

Mediators of the checkpoint-dependent inactivation of Cdc25 in human cells are excellent targets for generating therapeutics or therapeutic regimens that will enhance anti-cancer treatment, and reduce side-effects on normal cells.

To facilitate biochemical characterization of hCDS1, 6his-hCDS1 was expressed in insect cells, affinity purified and incubated in extracts of HeLa cells in the presence of an ATP-regenerating system. EDTA was added to inhibit kinase in the extract, and the rate of dephosphorylation and activation of Cdc2/CclinB was monitored.

Briefly, recombinant viruses encoding for 6his-hCDS1, 6his-Chkl, 6his-Cdc2 and GST-Cdc25C were generated using the Bac-to-Bac expression system from Gibco/BRL. 6his-fusion proteins were purified following the procedure described in Kumagai et al., (1995), Mol. Biol. Cell, 6: 199-213. GSH-sepharose beads were incubated for 15 minutes in Sf9 extracts; beads were collected by centrifugation and washed three-times with lysis buffer (50 mM Tris pH 8.0, 5 mM EDTA, 150 mM NaC1, 0.1% NP40, 5% glycerol, 0.1%3 -mercaptoethanol and protease inhibitors).

Beads were washed three-times with kinase assay buffer (50 mM Tris pH 7.4, 10 mM MgCl,) prior to phosphorylation reactions or three-times with phosphatase assay buffer mM imidazole pH 7.4, 5 mM EDTA and 0.1% 3-mercaptoethanol) prior to phosphatase assays.

Both 6his-Chkl and 6his-hCDSl were found to significantly reduce the activation of Cdc2/Cyclin B in these assays. The reduced activation of Cdc2 was dose dependent and required ATP. Confirmation that Cdc2 was not irreversibly inhibited by 6his-Chkl or 6his-hCDS was shown by the activation that resulted when excess was added after kinase treatment. Thus, both 6his-hCDS1 and 6his-Chkl can mimic the radiation-induced down-regulation of Cdc25 seen in extracts. These experiments used HeLa cell lysates that had been clarified by centrifugation, therefore it is unlikely that changes in sub-cellular locale could account for inactivation of (Peng et al., 1997, Science, 277: 1501-1505).

Example 5 Direct Effect of hCDS1 on Indirect mechanisms of inhibition of Cdc25 by hCDS1 could not be excluded by the cell lysate assays, therefore, affinity-purified reagents were used to determine direct phosphorylation and inhibition of GST-Cdc25 activity by hCDSI.

was incubated with either 6his-hCDS1, mock beads, or 6his-Chkl in the presence of y 32 P ATP for minutes at 30 0 C. Proteins were resolved by SDS-PAGE and visualized by autoradiography. GST-Cdc25 was phosphorylated by 6his-Chkl and by 6his-hCDSl. Assays were performed to determine if Cdc25 phosphatase activity was effected by this phosphorylation.

was assayed for its ability to activate the histone-H1 kinase activity of Cdc2/Cyclin B immuneprecipitates. It was found that phosphorylation of GSTby 6his-hCDS1 inhibited the ability of GST-Cdc25 to activate Cdc2/Cyclin B. Thus, these data demonstrate that 6his-hCDSl inactivated Cdc25 in vitro, and that Cdc25 is inactivated in vivo following DNA damage.

Since 6his-Chkl associates with GST-Cdc25 and has histone-Hl kinase activity in vitro (Sanchez et al., 1997, Science, 277: 1497-1501), analysis of Cdc2/Cyclin B kinase activity was obscured. In order to test GST-Chkl effects, an assay was used in which Cdc2 dephosphorylation was monitored by the disappearance of the slower migrating species of Cdc2 on gel-mobility analysis.

Briefly, phosphorylated Cdc2 was purified from Sf9 cells that had been simultaneously infected with recombinant baculoviruses encoding 6his-Cdc2, 6his-Weel, 6his-Mytl and GST-Cyclin B (Parker et al., 1992, Science, 257: 1955-1957. The 6his-Cdc2 complexed to Cyclin B was purified using GSH beads under the conditions for GSTexcept that 1 mM V04 was included in the lysis buffer. Western Blot analysis showed that quadruple infection resulted in phosphorylation of the majority of Cdc2/GST-Cyclin B at one or both inhibitory sites. These phosphatase assays were carried out in the presence of mM EDTA, and the absence of ATP, conditions that eliminate the possibility of 6his-Chkl phosphorylating Cdc2 or Cyclin B directly. GST-Cdc25 catalyses a reduction in the slower migrating phosphorylated forms of Cdc2. Prior phosphorylation of GST-Cdc25 by 6his-Chkl leads to a dosedependent reduction in GST-Cdc25 activity. These data confirm that Chkl negatively regulated Cdc25 activity (Furnari et al., 1997, Science, 277: 1495-1497; Weinert, 1997, Science, 277: 1450), and extend them by demonstrating that the negative regulation involves inactivation of the phosphatase activity.

Example 6 DNA Damage and Modification of hCDS1 As the previous data had shown that 6his-hCDS1 inactivates Cdc25, and that DNA damage is associated with inactivation, an assay was performed to determine if DNA damage leads to any modification or activation of hCDS1. Antisera raised against 6his-hCDSl was used in immune-complex kinase assays using HeLa cell lysates. A weak signal corresponding to hCDSl was detected in the sample from asynchronous HeLa cells; increased phosphorylation of hCDS1 was seen following irradiation.

Briefly, antibodies to hCDS1 were generated by immunizing a rabbit with 6his-hCDS1 purified from Sf9 cells (Harlow et al., Antibodies (Cold Spring Harbor Laboratory Press, NY, 1988). The resulting antisera immune-precipitates an active kinase of the expected molecular weight from Sf9 cells infected with 6his-hCDSl virus, but not from uninfected Sf9 cells, or from other cells infected with 6his-Chkl virus.

The results were confirmed as being due to hCDS1 by re-precipitation of the protein band following denaturation in 4% SDS. The in vitro phosphorylation is most likely due to autophosphorylation, and the increased signal reflects an increase in activity following irradiation. The increase of in vitro phosphorylation of p64Cdsl suggests that, like RAD53 and DUN1, hCDS1 is modified in response to DNA damage.

The effect of arresting DNA synthesis on phosphorylation of p64Cdsl was examined by further assay.

The hCDS1 from replication arrested cells behaved exactly like the protein from asynchronous cultures; no significant increase in phosphorylation was seen in response to thymidine or other agents that block DNA replication. The increased phosphorylation of p64Cdsl was detected following irradiation of thymidine-arrested cells. The effect of damaging DNA in cells that are predominantly arrested outside S-phase was also tested.

Cells were cultured in the presence of nocodazole for hours prior to irradiation. Again, a weak but detectable signal was seen in the unirradiated sample. However, irradiation of nocodazole arrested cells led to increased phosphorylation.

These findings surprisingly contrast with the results found in yeast, where fission yeast Cdsl has been found to be activated in response to incompletely replicated DNA (Boddy et al., 1998, Science, 280: 909-12; Lindsay et al., 1998, Genes and Dev., 12: 382-95). The results here show a role for human Cdsl in the DNA damage checkpoint rather than the replication checkpoint as previously found in yeast.

Example 7 Drug Identification The Cdc25 assays described above are suitable for use in the identification of chemical agents that would modify the DNA damage checkpoint mediated by hCDS1 and either by enhanced or inhibited activity. Thus a typical screening assay would use similar conditions as described above, plus addition of a reagent to be tested.

Monitoring of the activity of the assay components, i.e.

detection of phosphorylation as described above, can be conducted in comparison to controlreactions to detect both enhanced and inhibited activity.

Clearly such assays are readily adaptable to mechanical/automated apparatus and detection. With the fundamental elements of the assay reactions being known, the assay is clearly suited for use in conjunction with automated high-throughput low-signal apparatus which may incorporate microscopic slide arrays, or cell-biochip arrays in conjunction with CCD detection devices and the use of a visible signal triggered by phosphorylation or other reaction to kinase activity.

Example 8 Therapeutic Use The characterization of hCDSI and the elucidation that the role for human Cdsl is in the DNA damage checkpoint rather than the replication checkpoint as found in yeast, allows for the adaptation of this knowledge to the preparation of pharmaceuticals, and therapeutic methods for acting as an adjunct to chemotherapy of cancer. In particular, pharmaceutical formulations of the present invention incorporating cDNA, RNA, antisense molecules, hCDSI protein, antibodies against hCDS1 protein, or other therapeutics corresponding to those identified in the assays of the invention, can be administered in conjunction with any suitable chemotherapy agent in order to act as an adjunct to the main action of the chemotherapy agent. For example, the use of anticancer drugs such as antimetabolite, antibiotics, alkylating agents, microtubule inhibitors, steroid hormones and their antagonists, and others, is generally directed against metabolic sites essential to cell replication. While ideally these drugs should intervene only with the cellular processes unique to malignant 23A cells, the currently available anticancer drugs affect all proliferating cells, both normal and malignant. Thus, current chemotherapy is hampered by a steep dose-response curve for both toxic and therapeutic effects. Therefore, co-administration of the hCDSl-based drugs of the present invention, and drugs identified by the hCDS1 assays of the present invention, with chemotherapeutic agents will allow for enhanced killing of malignant cells.

One mechanism for enhanced killing is effected by disabling the DNA damage checkpoint control of malignant cells, thus making the administration of DNA damaging chemotherapeutic agents more effective. The disabling of the DNA damage control checkpoint can be effected by modifying the hCDSl response, as demonstrated by the data above.

Thus, the co-administration of novel hCDS1 based therapeutics in combination with any one or more anticancer agent is contemplated by the present invention.

For example, normal dosages of the anticancer drugs Cytarabine, Fludarabine, 5-Fluorouracil, 6-Mercaptopurine, Methotrexate, 6-Thioguanine, Bleomycin, Dactinomycin, Daunorubicin, Doxorubicin, Idarubicin, Plicamycin, Carmustine, Iomustine, Cyclophosphamide, Ifosfamide, Mechloroethamine, Streptozotocin, Navelbine® Paclitaxel, Vinblastine, Vincristine, Asparaginase, Cisplatin, Carboplatin, Etoposide, Interferons, Procarbazine etc., can be administered with the appropriate amount of hCDSl based drug so as to a) alter the length of time of administration, b) alter the time between administrations, c) alter the efficacy of the chemotherapeutic agent on malignant cells, or d) alter the side-effects of the chemotherapeutic agent on normal cells. The effects of the co-administration of hCDS1 based drugs can be any one or combination of these effects in addition to others.

23B Typically, destruction of cancer cells by chemotherapeutic agents follows first-order kinetics, for a log kill effect. Thus, the co-administration of hCDS1based therapeutics would be designed to enhance the log kill effect. Typically, chemotherapeutic treatment protocols call for a combination of drugs which act at different steps in the metabolic pathway, thus enhancing killing while staying below toxic levels. Thus, the coadministration of hCDSI based therapeutics would ideally be in combination with such protocols, and improve efficacy thereof.

Ultimately, the most effective therapeutic methods would combine targeted administration of chemotherapeutic drugs and/or MDR (multidrug resistance) inhibiting agents, with hCDS 1 based therapeutics, to specifically target and eliminate malignant cells via the cells' own uncontrolled replication without DNA damage repair, and thus eventual cell death.

The foregoing discussion and examples are intended as illustrative of the present invention and are not to be taken as limiting. Still other variants within the spirit and scope of this invention are possible and will readily present themselves to those of skill in the art.

In the claims which follow and in the preceding description of the invention, except where the context requires otherwise due to express language or necessary implication, the word "comprise" or variations such as "comprises" or "comprising" is used in an inclusive sense, i.e. to specify the presence of the stated features but not to preclude the presence or addition of further features in various embodiments of the invention.

It is to be understood that a reference herein to a prior art document does not constitute an admission that the document forms part of the common general knowledge in the art in Australia or in any other country.

Claims (16)

1. 2. The method of claim 1, further comprising administering a DNA damaging chemotherapeutic agent. is 3. The method of claim 1, wherein the compound is administered by administering a pharmaceutical composition comprising the compound together with a pharmaceutically acceptable carrier, diluent or excipient therefor.
2. 4. The method of claim 3, wherein the composition. further comprises a DNA damnaging chemotherapeutic agent.
3. 5. A method of treating cancer or proliferative disease comprising administering a therapeutically effective amount of a substance obtained by screening candidate substances in a method which comprises: providing a protein comprising the amino acid sequence of SEQ ID NO.: 2 or a functional equivalent, wherein said flunctional equivalent comprises 2s conservative substitutions, deletions or insertions of the amino acid sequence of Figure 2 such that said protein comprises different amino acids than those of the amino acid sequence shown in Figure 2, yet which substitutions, deletions or insertions do not affect the activity of the protein having the sequence of Figure 2 (SEQ ID NO: as measured by the ability to interact in the human cell cycle COMS ID No: SBMI-06084301 Received by IP Australia: Time 09:29 Date 2007-01-31 31/01 '07 WED 09:19 FAX 61299255911GRFIHAC j02 GRIFFITH HACK Q 020 -26 0 checkpoint pathway, wherein the protein or equivalent of said protein exhibits a kinase activity; contacting a substrate for said protein with the protein Under conditions such that the kinase will act upon the substrate; bringing the protein and substrate into contact with a candidate substance; tfl measuring the degree of increase or decrease in the kinase activity of the protein; and eN selecting a candidate substance which provides a decrease or increase in said o kinase activity to thereby obtain the substance. Ni 10 6. The method of claim 5, fuirther comprising administering a DNA damaging chemotherapeutic agent,
4. 7. The method of claim 5, wherein the substance is administered by administering a pharmaceutical composition comprising the substance together with a pharmaceutically acceptable carrier, diluent or excipient therefor.
5. 8. The method of claim 7, wherein the composition further comprises a DNA damaging chemotherapeutic agent.
6. 9. A method of increasing susceptibility of cancer cells to chemotherapy and/or radiotherapy, which method comprises administering to a patient a therapeutically effective amount of a substance obtained by screening candidate substances in a method which comprises: providing a protein comprising the amino acid sequence of SEQ ID NO.: 2 or a functional equivalent, wherein said functional equivalent comprises conservative substitutions, deletions or insertions of the amino acid sequence of Figure 2 such that said protein comprises different amino acids than those of the amino acid sequence shown in Figure 2, yet which substitutions, deletions or insertions do not affect the activity of the protein having the sequence of Figure 2 (SEQ ID NO: as measured by the ability to interact in the human cell cycle checkpoint pathway, wherein the protein or equivalent of said protein exhibits a kinase activity; COMSID No: SBMI-060)84301 Received by IP Australia: Time 09:29 Date 2007-01-31 31/01 '07 WED 09:20 FAX 61299255911 GRIFFITH HACK IMo21 -27 0 0 contacting a substrate for said protein with the protein under conditions such that the kinase will act upon the substrate; bringing the protein and substrate into contact with a candidate substance; en measuring the degree of increase or decrease in the kinase activity of the S protein; and selecting a candidate substance which provides a decrease or increase in said in kinase activity to thereby obtain the substance. C. 10. A method of treating cancer or proliferative disease in a patient comprising administering a therapeutically effective amount of a compound o capable of modifying the activity of CDS 1 human cell cycle checkpoint pathway protein comprising the amino acid sequence depicted in Figure 2 (SEQ ID NO: to a patient in need of such treatment, wherein the compound is identified by a method which comprises combining Cdc25 protein with human hCDS 1 protein in the presence of ATP and the said compound, and detecting Cdc25 phosphatase activity to thereby obtain said compound.
7. 11. The method of claim 10, further comprising administering a DNA damaging chemotherapeutic agent.
8. 12. The method of claim 10, wherein the compound is administered by administering a pharmaceutical composition comprising the compound 2o together with a pharmaceutically acceptable carrier, diluent or excipient therefor.
9. 13. The method of claim 12, wherein the composition further comprises a DNA damaging chemotherapeutic agent.
10. 14. A method of treating cancer or proliferative disease in a patient comprising administering a substance obtained by screening candidate substances in a method which comprises combining a protein having the amino acid residue sequence shown in Figure 2 (SEQ ID NO.: 2) and Cdc25 protein under phosphorylating conditions, contacting the resulting combination with a candidate substance, and determining any change in Cdc25 protein phosphorylation activity in order to identify candidate substances which modulate phosphorylation activity, and selecting a candidate substance which COMS ID No: SBMI-06084301 Received by IP Australia: Time 09:29 Date 2007-01-31 31/01 '07 WED 09:20 FAX 61299255911GRFIhHC 02 GRIFFITH HACK 16022 -28 0 modulates phophorylation activity to thereby obtain the substance, to a patient in need thereof The method of claim 14, further comprising administering a DNA damaging chemotherapeutic agent. s 1. The method of claim 14, wherein the substance is administered IC) by administering a pharmaceutical composition comprising the substance together wit a pharmaceutically acceptable carrier, diluent or excipient therefor. ci17. The method of claim 16, wherein the composition fturther 0 comprises a DNA damaging chemotherapeutic agent.
11. 18. Use of a compound that is an inhibitor or activator of expression of the CDS1I human cell cycle checkpoint pathway protein comprising the amino acid sequence depicted in Figure 2 (SEQ ID NO: in the manufacture of a medicament for treating cancer or proliferative disease, wherein the compoundis identified by a method which comprises contacting a cell expressing the proteins of the CDS 1 human cell cycle checkpoint pathway with said compound, and comparing the level of expression of the CDS I human cell cycle checkpoint pathway protein of said cell against a ccll which has not been in contact with said compound, wherein said compound increases or decreases expression of the CDS 1 human cell cycle checkpoint pathway protein.
12. 19. Use of a compound that is an inhibitor or activator of expression of the CDS 1 human cell cycle checkpoint pathway protein comprising the amino acid sequence depicted in Figure 2 (SEQ ID NO: and a DNA damaging chemotherapeutic agent, in the manufacture of a medicament for treating cancer or proliferative disease, wherein the compound is identified by a method which 2S comprises contacting a cell expressing the proteins of the CDSlI human cell cycle checkpoint pathway with said compound, and comparing the level of expression of the CDS 1 human cell cycle checkpoint pathway protein of said cell against a cell which has not been in contact with said compound, wherein said compound increases or decreases expression of the CDS I human cell cycle checkpoint 3o pathway protein. COMS ID No: SBMI-06084301 Received by IP Australia: Time 09:29 Date 2007-01-31 31/01 '07 WED 09:20 FAX 61299255911GRFIHAC j03 GRIFFITH HACK [a 023 -29- Use of a substance obtained by screening candidate substances in a method which comprises: providing a protein comprising the amino acid sequence of SEQ ID NO.: 2 or en a functional equivalent, wherein said functional equivalent comprises conservative substitutions, deletions or insertions of the amino acid sequence of Figure 2 such that said protein comprises different amino acids than those of the amino acid sequence shown in Figure 2, yet which substitutions, deletions or en insertions do not affect the activity of the protein having the sequence of Figure 2 o (SEQ ID NO: as measured by the ability to interact in the human cell cycle checkpoint pathway, wherein the protein or equivalent of said protein exhibits a kinase activity; contacting a substrate for said protein with the protein under conditions such that the kinase will act upon the substrate; bringing the protein and substrate into contact with a candidate substance; measuring the degree of increase or decrease in the kinase activity of the protein; and selecting a candidate substance which provides a decrease or increase in said kinase activity to thereby obtain the substance, in the manufacture of a medicament for treating cancer or proliferative disease.
13. 21. Use of a substance obtained by screening candidate substances in a method which comprises: providing a protein comprising the amino acid sequence of SEQ ID NO.: 2 or a functional equivalent, wherein said functional equivalent compnises conservative substitutions, deletions or insertions of the amino acid sequence of Figure 2 such that said protein comprises different amino acids thani those of the amino acid sequence shown in Figure 2, yet which substitutions, deletions or insertions do not affect the activity of the protein having the sequence of Figure 2 (SEQ ID NO: as measured by the ability to interact in the human cell cycle checkpoint pathway, wherein the protein or equivalent of said protein exhibits a 3o kinase activity; COMS ID No: SSMI-06084301 Received by IP Australia: Time 09:29 Date 2007-01-31 31/01 '07 WED 09:21 FAX 61299255911GRFIhHC 102 GRIFFITH HACK IM 024 o contacting a substrate for said protein with the protein under conditions such that the kinase will act upon the substrate; bringing the protein and substrate into contact with a candidate substance; en measuring the degree of increase or decrease in the kinase activity of the protein; and selecting a candidate substance which provides a decrease or increase in said kinase activity to thereby obtain the substance, en and a DNA damaging ehemotherapeutic agent, o in the manufacture of a mnedicament for treating cancer or proliferative disease.
14. 22. Use of a substance obtained by screening candidate substances in a method which comprises: providing a protein comprising the amino acid sequence of SEQ ID NO.: 2 or a fuinctional equivalent, wherein said furnctianal equivalent comprises conservative substitutions, deletions or insertions of the amino acid sequence of Figure 2 such that said protein comprises different amino acids than those of the amino acid sequence shown in Figure 2, yet which substitutions, deletions or insertions do not affect the activity of the protein having the sequence of Figure 2 (SEQ ID NO: as measured by the ability to interact in the humnan cell cycle checkpoint pathway, wherein the protein or equivalent of said protein exhibits a kinase activity; contacting a substrate for said protein with the protein under conditions such that the kinase will act upon the substrate; bringing the protein and substrate into contact with a candidate substance; measuring the degree of increase or decrease in the kinase activity of the protein; and selecting a candidate substance which provides a decrease or increase in said kinase activity to thereby obtain the substance, in the manufacture of a medicament for increasing susceptibility of cancer cells to chemotherapy and/or radiotherapy. COMS ID No: SBMI-06084301 Received by IP Australia: Time 09:29 Date 2007-01-31 31/01 '07 WED 09:21 FAX 61299255911 GRIFFITH HACK M 025 31 o 23. Use of a compound capable of modifying the activity of CDS1 human cell cycle checkpoint pathway protein comprising the amino acid sequence depicted in Figure 2 (SEQ ID NO: in the manufacture of a en medicament for treating cancer or proliferative disease in a patient, wherein the compound is identified by a method which comprises combining Cdc25 protein with human hCDS 1 protein in the presence of ATP and the said compound, and 0 detecting Cdc25 phosphatase activity to thereby obtain said compound. C 24. Use of a compound capable of modifying the activity of CDS 1 0 human cell cycle checkpoint pathway protein comprising the amino acid C 10 sequence depicted in Figure 2 (SEQ ID NO: and a DNA damaging chemotherapeutic agent, in the manufacture of a medicament for treating cancer or proliferative disease in a patient, wherein the compound is identified by a method which comprises combining Cdc25 protein with human hCDS 1 protein in the presence of ATP and the said compound, and detecting Cdc25 phosphatase activity to thereby obtain said compound. Use of a substance obtained by screening candidate substances in a method which comprises combining a protein having the amino acid residue sequence shown in Figure 2 (SEQ ID NO.: 2) and Cdc25 protein under phosphorylating conditions, contacting the resulting combination with a 2 candidate substance, and determining any change in Cdc25 protein phosphorylation activity in order to identify candidate substances which modulate phosphorylation activity, and selecting a candidate substance which modulates phosphorylation activity to thereby obtain the substance, in the manufacture of a medicament for treating cancer or proliferative disease in a patient.
15. 26. Use of a substance obtained by screening candidate substances in a method which comprises combining a protein having the amino acid residue sequence shown in Figure 2 (SEQ ID NO,; 2) and Cdc25 protein under phosphorylating conditions, contacting the resulting combination with a 3 candidate substance, and determining any change in Cdc25 protein COMS ID No: SBMI-06084301 Received by IP Australia: Time 09:29 Date 2007-01-31 20/02 '07 TUE 15:26 FAX 61299255911 GRIFFITH HACK 1004 32 phosphorylation activity in order to identify candidate substances which modulate phosphorylation activity, and selecting a candidate substance which modulates phosphorylation activity to thereby obtain the substance, and a DNA CN damaging chemotherapeutic agent, in the manufacture of a medicament for treating cancer or proliferative disease in a patient.
16. 27. A method according to any one of claims 1, 5, 9, 10 or 14, substantially as hereinbefore described with reference to any one of the e,1 Examples. o 28. Use according to any one of claims 18 to 26, substanti ily as o hereinbefore described with reference to any one of the Examples. COMS ID No: SBMI-06315604 Received by IP Australia: Time 15:29 Date 2007-02-20