WO2001009373A2 - Native cdc25 substrates, compositions and uses related thereto - Google Patents

Abstract

The present invention relates to the preparation and use of a natural substrate for human Cdc25 which is useful for the direct measurement of Cdc25 activity in a quantifiable and reproducible manner.

Description

Native CDC25 Substrates, Compositions and Uses Related Thereto

1. Background of the Invention

The progression of a proliferating eukaryotic cell through the cell-cycle checkpoints is controlled by an array of regulatory proteins that guarantee that mitosis occurs at the appropπate time Protein phosphorylation is the most common post-translational modification that regulates processes inside the cells, and a large number of cell cycle transitions are regulated by the phosphorylation states of vaπous proteins In particular, the execution of vaπous stages of the cell-cycle is generally believed to be under the control of a large number of mutually antagonistic kinases and phosphatases A paradigm for these controls is the Cdc2 protein kinase, a cyclm-dependent kinase (Cdk) whose activity is required for the tπggeπng of mitosis in eukaryotic cells (for reviews, see Hunt (1989) Curr Opin Cell Biol 1.268-274, Lewin (1990) Cell 61 743-752, and Nurse (1990) Nature 344:503-508) Duπng mitosis, the Cdc2 kinase appears to tπgger a cascade of downstream mitotic phenomena such as metaphase alignment of chromosomes, segregation of sister chromatids in anaphase, and cleavage furrow formation Many target proteins involved in mitotic entry of the proliferating cell are directly phosphorylated by the Cdc2 kinase For instance, the Cdc2 protein kinase acts by phosphorylatmg a wide vaπety of mitotic substrates involved in regulating the cytoskeleton of cells, such that entry into mitosis is coordinated with dramatic rearrangement of cytoskeletal elements

The Cdks are subject to multiple levels of control One well-characteπzed mechanism regulating the activity of Cdc2, the best-studied Cdk2, involves the phosphorylation of tyrosme, threonme, and seπne residues; the phosphorylation level of which vanes duπng the cell-cycle (Krekk et al. (1991) EMBO J. 10.305-316; Draetta et al (1988) Nature 336 738- 744; Dunphy et al. (1989) Cell 58:181-191; Morla et /. (1989) Cell 58:193-203, Gould et al (1989) Nature 342:39-45; and Solomon et al (1990) Cell 63:1013-1024). The phosphorylation of Cdc2 on Y15 and T14, two residues located the putative ATP binding site of the kinase, negatively regulates kinase activity This inhibitory phosphorylation of Cdc2 is mediated at least in part by the Weel and Mikl tyrosme kinases (Russel et al (1987) Cell 49:559-567; Lundgren et αZ (1991) Cell 64:1111-1122; Featherstone et al (1991) Nature 349:808-811; and Parker et al. (1992) PNAS 89:2917-2921). Mytl is the threonme kmase responsible for the phosphorylation of T14 (Mueller et al (1995) Science 270:86-89) These kinases act as mitotic inhibitors, over-expression of which causes cells to arrest in the G2 phase of the cell-cycle. By contrast, loss of function of Weel causes a modest advancement of mitosis, whereas loss of both Weel and Mikl function causes grossly premature mitosis, uncoupled from all checkpoints that normally restrain cell division (Lundgren et al. (1991) Cell 64: 1111-1122). As the cell is about to reach the end of G2, dephosphorylaUon of the Cdc2-ιnactιvatιng T14 and Y15 residues occurs leading to activation of the Cdc2 complex as a kinase. A stimulatory phosphatase, known as Cdc25, is responsible for Tyr-15 and Thr-14 dephosphorylation and serves as a rate-limiting mitotic activator. (Dunphy et al. (1991) Cell 67:189-196; Lee et al. (1992) Mol. Biol Cell. 3:73-84; Millar et al. (1991) EMBO J 10.4301-4309; and Russell et al. (1986) Cell 45 145-153)

2. Summary of the Invention

The present invention relates to the preparation and use of a natural substrate for human Cdc25 which is useful for the direct measurement of Cdc25 activity in a quantifiable and reproducible manner. The natural substrate, a human Cdk/Cyc complex subjected to inhibitory phosphorylation by Xenopus Mytl ox Xenopus Weel, is an excellent substrate only for Cdc25 dual specificity phosphatases and is essentially inert to other phosphatases. The activity of Cdc25 with its natural substrate is 10⁵-fold higher compared to phosphopeptides and directed mainly at phosphothreonine, not phosphotyrosme as is the case for phosphopeptides The detection of phosphothreonine and/or phosphotyrosme hydrolysis in this natural substrate assay allows the detection and quantifiable analysis of highly specific inhibitors of the Cdc25 reaction.

In one aspect, the present invention provides a method for identifying a compound which is an inhibitor of mammalian Cdc25 phosphatase activity, which assay includes the steps of a) combining, in a reaction mixture, a Cdc25 phosphatase, phosphorylated complexes of a phosphorylated Cdk/cyclm complex, and a test compound, and b) detecting threonme-14 (T14) and/or tyrosme-15 (Y15) dephosphorylation of the phosphorylated Cdk/cyclm complex, wherein the test compound is an inhibitor of the Cdc25 phosphatase if it decreases the ability of the Cdc25 phosphatase to dephosphorylate the phosphorylated Cdk/cyclin complex; wherein (1) the Cdc25 phosphatase will dephosphorylate the phosphorylated Cdk/cyclm complex in the absence of an inhibitor of the phosphatase, and (n) the reaction mixture is defined with respect to phosphorylation state of the Cdk/cyclm complex, or is substantially free of Cdk/cyclm associated proteins, or both.

In preferred embodiments, the Cdk/cyclm complex is composed of human Cdk and cyc n proteins, or portions thereof sufficient to form the complex and serve as a substrate for the Cdc25 phosphatase. The complex can include separate Cdk and cychn polypeptides, or can be a fusion protein including the two subunit polypeptide chains. In prefeπed embodiments, the Cdk/cyclin complex is a human Cdk2/CycA complex.

In certain embodiments, Thr-161 of the Cdk protein can be phosphorylated. For instance, a heterologous source of Mytl can be used to phosphorylate the Cdk/cyclin complex. For instance, the Mytl can be from a cross-species source such as, but not limited to, Xenopus, starfish, yeast, or Drosophila.

In certain embodiments, Weel can be used to phosphorylate the Cdk/cyclin complex, e.g., wherein the Weel is derived from a different cross-species source such as but not limited to starfish, yeast, or Drosophila.

In prefeπed embodiments, the Cdk/cyclin complex includes less than 25 percent (w/w protein) of pl3^{suc l}, INK4 or WAFl/Cipl proteins.

In preferred embodiments, the Cdk/cyclin complex can be a Cdk2/CycA, Cdc2/CycB, Cdk2/CycE, Cdk4/CycD, or Cdk6/CycD complex.

In prefeπed embodiments, the Cdc25 phosphatase is a human Cdc25 phosphatase, and more preferably a human Cdc25A phosphatase. For example, the Cdc25 phosphatase can include a polypeptide designated by SEQ ID Nos. 2, 4 or 6, or a catalytic fragment thereof, or which is at least 70, 80, 90 or 95 percent identical to one of those sequence listings. In prefeπed embodiments, the Cdc25 phosphatase includes an amino acid sequence which is encoded by a nucleic acid which hybridizes under stringent conditions to SEQ ID Nos 1 , 3 or 5.

In certain embodiments of the subject assay, dephosphorylation of the phosphorylated Cdk2/CycA complex is determined by detecting the release of free inorganic phosphate (3lp 32p _or 33p) ι_n other embodiments, disruption of complex formation between the phosphorylated Cdk2/CycA complex and a catalytically inactive mutant of Cdc25 is used to evaluate the test compounds.

In addition to preparations of the Cdk/cyclin complexes, another aspect of the present invention relates to methods for purifying Cdk/cyclin complexes substantially free of contaminants, e.g., Cdk/cyclin associated proteins. By way of example, the present invention provides protein compositions including cyclin/cdk protein complexes, but having less than 25 percent (w/w) of contaminating proteins, more preferably less than 20, 10, 5 or even 2 percent of contaminating protein.

As described herein, a unique feature of the Cdk/cyclin preparations of the present invention is the fact that a defined amount of phosphorylation can be attained in the preparation. In prefeπed embodiments, at least 10 percent of the Cdk/cyclin complex in the preparation is phosphorylated on T14 and/or Y15, and more preferably at least 25 or 50 percent. In certain embodiments, it will be desirable that the ratio of phosphorylated complex to unphosphorylated complex in the Cdk/cyclin preparation is high. In prefeπed embodiments, at least 80% of the Cdk/cyclin complexes present in the composition are phosphorylated, more preferably at least 85%, 90% or even 95% of the Cdk/cyclin complex are phosphorylated.

In certain prefeπed embodiments, the composition is relatively homogenous with respect to the cdk and cyclin polypeptides making up the complexes of the composition, or at least the phosphorylated forms. In prefeπed embodiments, at least 80% of the Cdk/cyclin complexes of the subject compositions include the same cdk and cyclin polypeptide, more preferably at least 85%, 90% or even 95% of the Cdk/cyclin complex are phosphorylated.

The present invention also provides various means for preparing substrates having both the desired purity and homogenity of phosphorylation state, i.e., the bis- and mono- phosphorylated substrate can be produced by a process which ensures integrity and homogeneity. Accordingly, in one embodiment, this invention provides compositions comprising Cdk/cyclin complexes, wherein the phosphorylated forms of the complex which are present in the composition are substantially homogenous with respect to phosphorylation at T14 and/or Y15. In one aspect, at least 75% of the complexes are bis-phosphorylated, more preferably, at least 85% of the complexes are bis-phosphorylated, and even more preferably at least 95% of the complexes are bis-phosphorylated.

In still other embodiments, the phosphorylated Cdk/cyclin complexes of the composition are substantially homogenous for mono-phosphorylated Cdk, e.g., phosphorylated on only one of T14 or Y15. In prefeπed embodiments, at least 75% of the complexes are mono-phosphorylated, more preferably at least 85% of the complexes are mono-phosphorylated, and even more preferable at least 95% of the complexes are mono- phosphorylated.

As discussed above, the Cdk/cyclin complexes of this invention includes cdks and cyclins which may be either full length or may be fragments wherein the fragments retain the ability to form Cdk/cyclin complexes. In one embodiment, this invention provides bis- phosphorylated substrates of cdc25; examples include bis-phosphorylated Cdk2/cyclin A, Cdk2/cyclin E, Cdk2/cyclin B, Cdk4/cyclin A, cdk6/cyclin A, and Cdkl/cyclin B. This invention also provides a multi-step process for preparing these natural substrates.

The scope of the invention includes equivalent substrates, specifically, equivalent substrates broadly include the various mono- and bis phosphorylated cdc25 substrates such as mono- and bis phosphorylated Cdk2/cyclinA, Cdk4/cyclinA, cdkό/cyclin A, Cdk2/cyclin E, Cdk2/cyclin B, and Cdkl/cyclin B. This term includes both the bis-phosphorylated and mono-phosphorylated substrates. Equivalents include substrate complexes which will mimic (agonize) the action of the natural substrates or which will antagonize the actions of the natural substrate.

3. Description of the Drawings

Figure 1.1

Autoradiography following SDS-PAGE of a reaction mixture containing 1 μg Xenopus GST-Mytl and 3.3 mM γ-³²P-ATP in the presence (lane 1) or absence (lane 2) of 3 μg of human Cdk2/CycA demonstrating that Mytl undergoes self-phosphorylation as well as phosphorylating Cdk2.

Figure 1.2

LC-MS of the Cdk2 part of the human Cdk2/CycA complex before and after phosphorylation with Xenopus Mytl showing the addition of two phosphates (= 160 Da) to the Cdk2 subunit.

Figure 1.3

Autoradiography of Mytl -phosphorylated Cdk2/CycA following phosphoaminoacid analysis showing co-migration of the radiolabeled phosphothreonine and phosphotyrosine with the standards indicated by the dotted lines.

Figure 1.4

Autoradiography following SDS-PAGE of a reaction mixture containing Myt- phosphorylated Cdk2/CycA following no addition (lane 1), treatment with Cdc25A (lane 2) or treatment with Cdc25A(C430S) indicating that the Mytl -phosphorylated complex is a substrate for active Cdc25A but not for its active site mutant.

Figure 2.1

The reaction time-course of the Cdc25A-catalyzed dephosphorylation of Cdk2- pTpY/CycA shows biphasic kinetics as shown more clearly in the inset. Reactions contained 100 nM Cdk2-pTpY/CycA phosphorylated by Xenopus Mytl in the presence of γ-³²P-ATP and 11 nM GST-Cdc25A. The reactions were quenched with TCA at varying time points and the supernatants of the samples were subjected to scintillation counting following centrifugation of the precipitated protein containing unreacted starting material.

Figure 2.2

Comparative phosphatase activity measurements of various phosphatases using the substrate Cdk2-pTpY/CycA demonstrates that Cdc25A has the highest activity. Cdc25C is approximately 100-fold less active than Cdc25A whereas the phosphatases VHR and Ptplb are 10⁴ to 10⁵-fold less active toward Cdk2-pTpY/CycA. Reaction mixtures contained 100 nM Cdk2-pTpY/CycA phosphorylated by Xenopus Mytl in the presence of γ-³²P-ATP and varying amounts of the phosphatases. the reactions were quenched with TCA after 30 min at room temperature and the supematants of the samples were subjected to scintillation counting following centrifugation of the precipitated protein containing unreacted starting material.

Figure 2.3

Injection of various Cdc25A constructs into Xenopus oocytes triggers Germinal Vesicle Breakdown at varying concentrations of Cdc25A dependent on the construct injected. Full-length Cdc25A is approximately 10-fold more potent than the catalytic domain Cdc25(337-524), whereas the even shorter catalytic domain Cdc25(337-504) has no apparent activity in this assay.

Figure 2.4

Autoradiography of Cdc25-treated Cdk2-pTpY/CycA following phosphoaminoacid analysis showing a progressive reduction of the intensity of the pT spot compared to the pY spot over the time-course of the reaction, as indicated. The timepoints coπespond to the samples in Figure 2.1. The co-migration of the radiolabeled phosphothreonine and phosphotyrosine with the standards are indicated by the dotted lines.

Figure 3.1

IC-50 determinations for tungstate on Cdc25A(324-337) against the substrate mFP and the substrate Cdk2-pTpY/CycA give identical values. Reactions contained varying concentrations of tungstate (2000 to 1 μM), 5 nM Cdc25(324-337), and either 5 μM mFP or 100 nM Cdk2-pTpY/CycA.

Figure 3.2

IC-50 determinations for p27(Kipl) on Cdc25A(324-337) against the substrate mFP and the substrate Cdk2-pTpY/CycA give very different results. Reactions contained varying concentrations of p27(Kipl) (3000 to 1 μM), 5 nM Cdc25(324-337), and either 5 μM mFP or 100 nM Cdk2-pTpY/CycA.

4. Detailed Description of the Invention

4.1 General

The eukaryotic cell cycle is tightly regulated by the action of the cyclin-dependent kinases (Cdks). The kinase activity of these complexes is directly responsible for the initiation and progression of successive phases of the cell cycle by modification of proteins required for the activation of genes involved in DNA synthesis and in the structural reorganization of the cell during mitosis. Therefore, reversible regulation of Cdk kinase activity is key to controlling the cell cycle. Monomeric Cdks are generally, un- phosphorylated and inactive, and association with the appropriate cyclin partner to form a heterodimeric complex (i.e. Cdc2/CycB, Cdk2/CycA) is the first step required for kinase activity. The kinase activity of Cdk/cyclin complexes is also regulated, at one level, by phosphorylation. Phosphorylation of the conserved threonine (T14) and tyrosine residues (T15) inhibits the kinase activity. The activity of the complex is therefore regulated by a set of antagonist kinases (e.g. Weel, Mytl) and phosphatase (Cdc25). Thus the phosphorylation (by Weel and Mytl) and the dephosphorylation (by Cdc25s) of T4 and Y15 in Cdk/Cyclin complexes creates a fine balance which controls cell cycle progression. Phosphorylation of T160/T161 by the Cdk-activating kinase (CAK) also regulates the kinase activity of the complex.

While it was known that Cdc25 phosphatases play a vital role in cell cycle control, the details of their substrate specificity and catalytic mechanism of action have remained somewhat elusive. In particular, the lack of availability of a purified, homogenous source of the natural substrates has been a problem in the art which has hindered detailed biochemical investigations of the Cdc25 phosphatase enzymes. This, in turn, has limited the scope of assays designed to identify compounds which are selective inhibitors of these phosphatases.

The investigation of the mechanism and the regulation of Cdc25 has long been hampered by the lack of availability of suitable amounts of purified natural substrates. Drug- screening and characterization for antagonists of Cdc25 has also been limited by the absence of natural substrate screens and assays. Historically, substrates for Cdc25 have been prepared to yield a non-homogeneous or non-soluble substrate and the activity of these substrates has been measured mostly by indirect assays. Originally, pl3sucl (or the human analog p9Cks) affinity chromatography was used to extract Cdc2/CycB from Xenopus oocytes (Jessus et al. (1990) FEBS Lett. 266:4-8); Kumagai & Dunphy (1991) Cell 64:903-914; Borgne & Meijer (1996) J. Biol. Chem. 271:27847-27854). This procedure isolates a Sepharose or agarose bead-bound complex of Cdc2/CycB with an undefined phosphorylation state complexed to pl3sucl, a protein whose function as a tight-binder of Cdc2 is still undefined. A variety of "in vitro bathing protocols have been developed to prepare Cdc25 substrates. For example, insect cells were used to produce tagged CycB which was then incubated in activated oocyte lysates and reisolated as a phosphorylated complex by immunoprecipitation via the CycB tag (Kumagai & Dunphy (1992) Cell 170:139-151). Modifications of this method include co- expression of the kinase and the cyclin prior to the "bathing" procedure as well as the use of His tags (Kumagai & Dunphy (1995) Molec. Biol. Cell 6:199-213). All of these procedures potentially yield variable phosphorylation states of the cyclin and/or kinase (i.e. T14, Y15, T160/161), co-purify Cdk/Cyclin-binding proteins (i.e. pl3sucl or members of the INK4 or WAFl/Cipl family of inhibitors), or generate insoluble (i.e. bead-bound) substrates for Cdc25.

These procedures do not yield quantifiable amounts of material usable for determination of kinetic constants or for performing high-throughput screening. Because of inadequate homogeneity in phosphorylation, a lack of quantitative amounts of the pure substrate, and contamination by other Cdk/cyclin associated proteins, such preparations have been, at best, only marginally useful for providing adequate substrate to use in screening assays or kinetic/mechanism studies. The lack of homogeneity in the phosphorylation state of Thr-14 and Tyr-15, for example, can confound results due to the differences in the kinetics of the dephosphorylation of those residues. In addition, other proteins which may co-purify with the prior art cdk-cyclin preparations , such as p21^cΦ¹ or pl6^ink4, are inhibitors of Cdk activity and can thus affect the catalytic activity of Cdk/cyclin complexes.

As described in the appended examples, we have succeeded in preparing defined preparations of the native substrate, i.e., with respect to cyclin and cdk content and/or with respect to mono- and/or bis-phosphorylated forms of Cdk/cyclin complexes. For example, the subject invention provides compositions including, as a homogenous preparation, defined Cdk/cyclin complexes, e.g., which are relatively pure with respect to Cdk/cyclin associated proteins (substantially lacking, for example, pl3^sucl, Ink4 and Wafl/Cipl proteins). The subject invention also provides compositions of Cdk/cyclin complexes which are homogeneous preparations of a particular phosphorylated complex, i.e., mono- or bis- phosphorylated Cdk/cyclin complexes e.g., mono- or bis-phosphorylated complexes of Cdk2/cyclin A, Cdk2/cyclin E, Cdk2/cyclin B, Cdk4/cyclin A, and Cdkl/cyclin B to name a few. In other embodiments, the invention provides methods of preparing these natural substrates, which methods ensure the integrity and homogeneity of these substrates.

The present invention also provides drug screening assays which ascertain if a test compound can inhibit the ability of a Cdc25 phosphatase to bind and/or dephosphorylate its native substrate, e.g., a phosphorylated Cdk/Cyclin complex. As described below, we have also unexpectedly found that the dephosphorylation of the complex is biphasic in nature, e.g. dephosphorylation of the phosphothreonine (T14) and the phosphotyrosine (Y15) occur at different rates. For instance, it was found that dephosphorylation of the phosphotyrosine reaction is on the order of a hundred times (lOOx) slower than the phosphothreonine dephosphorylation reaction. This sequential dephosphorylation kinetics provides a basis for identifying inhibitors which can discriminate between the reactions of each phase, i.e, inhibitors which are more selective for one of either of T14 dephosphorylation or Y15 dephosphorylation reactions.

4.2 Definitions Before further description of the invention, certain terms employed in the specification, examples, and appended claims are collected here for convenience.

As used herein, the term "nucleic acid" refers to polynucleotides such as deoxyribonucleic acid (DNA), and, where appropriate, ribonucleic acid (RNA). The term should also be understood to include, as equivalents, analogs of either RNA or DNA made from nucleotide analogs, and, as applicable to the embodiment being described, single- stranded (such as sense or antisense) and double-stranded polynucleotides.

A "Cdk/cyclin" complex, as used herein, includes complexes which are formed between cyclin dependent kinases and cyclins, the cyclins and cdks may be either full length or may be fragments, wherein said fragment specifically (1) retain their ability to interact and form Cdk/cyclin complexes; and (2) in the case of cdk polypeptides other than Cdk4 and Cdk6, one or both of the threonine-14 (T14) and tyrosine-15 (Y15) residues, or the equivalent thereof, are preserved in the fragments. In the case of Cdk4 and Cdk 6, the residue coπesponding to T14 is an alanine, and the residue coπesponding to Y15 is Y17 and Y24, respectively.

As used herein, the term "bis-phosphorylated" Cdk/cyclin complexes include those complexes wherein both T14 and Y15 are phosphorylated; whereas, "mono-phosphorylated" complexes include those complexes wherein only either T14 or Y15 is phosphorylated. T160/T161, and/or other residues, may be phosphorylated or unphosphorylated in these bis- and mono phosphorylated complexes.

The term "full inactivation phosphorylation" refers to bis-phosphorylated forms of Cdks, e.g., wherein both T14 and Y15 are phosphorylated, or, in the case of Cdk4 and Cdk6 (and others which lack one of the two threonine or tyrosine residue) monophosphorylated Y15/Y17.

As used herein, the term "Xenopus Mytl" and "Xenopus Weel" refer to the kinases as derived from the organism Xenopus laevis.

Unless otherwise evident from the context, the term "phosphorylated complex" refers to either mono- or bis-phosphorylated Cdk cyclin complexes.

As used herein, "substantially lacking" of a particular protein or proteins means that the preparation includes less than 25 percent (M/M ratio) of that protein(s) as a contaminant, and more preferably less than 20, 10, 5 or 2 percent of that protein(s) as a contaminant.

As used herein, the term "gene" or "recombinant gene" refers to a nucleic acid comprising an open reading frame encoding a polypeptide of the present invention, including both exon and (optionally) intron sequences. A "recombinant gene" refers to nucleic acid encoding such regulatory polypeptides, which may optionally include intron sequences which are derived from chromosomal DNA. The term "intron" refers to a DNA sequence present in a given gene which is not translated into protein and is generally found between exons. As used herein, the term "transfection" means the introduction of a nucleic acid, e.g., an expression vector, into a recipient cell by nucleic acid-mediated gene transfer.

The term "purified" as used herein preferably means at least 80% by dry weight, more preferably in the range of 95-99% by weight, and most preferably at least 99.8% by weight, of biological macromolecules of the same type present (but water, buffers, and other small molecules, especially molecules having a molecular weight of less than 5000, can be present). The term "pure" as used herein preferably has the same numerical limits as "purified" immediately above. "Isolated" and "purified" do not encompass either natural materials in their native state or natural materials that have been separated into components (e.g., in an acrylamide gel) but not obtained either as pure (e.g. lacking contaminating proteins, or chromatography reagents such as denaturing agents and polymers, e.g. acrylamide or agarose) substances or solutions.

As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of prefeπed vector is an episome, i.e., a nucleic acid capable of extra-chromosomal replication. Prefeπed vectors are those capable of autonomous replication and/expression of nucleic acids to which they are linked. Vectors capable of directing the expression of genes to which they are operatively linked are refeπed to herein as "expression vectors". In general, expression vectors of utility in recombinant DNA techniques are often in the form of "plasmids" which refer to circular double stranded DNA loops which, in their vector form are not bound to the chromosome. In the present specification, "plasmid" and "vector" are used interchangeably as the plasmid is the most commonly used form of vector. However, the invention is intended to include such other forms of expression vectors which serve equivalent functions and which become known in the art subsequently hereto.

"Transcriptional regulatory sequence" is a generic term used throughout the specification to refer to DNA sequences, such as initiation signals, enhancers, and promoters, which induce or control transcription of protein coding sequences with which they are operably linked. In prefeπed embodiments, transcription of a recombinant gene is under the control of a promoter sequence (or other transcriptional regulatory sequence) which controls the expression of the recombinant gene in a cell-type in which expression is intended. It will also be understood that the recombinant gene can be under the control of transcriptional regulatory sequences which are the same or which are different from those sequences which control transcription of the naturally-occurring form of the protein.

"Homology" refers to sequence similarity between two peptides or between two nucleic acid molecules. Homology can be determined by comparing a position in each sequence which may be aligned for purposes of comparison. When a position in the compared sequence is occupied by the same base or amino acid, then the molecules are homologous at that position. A degree of homology between sequences is a function of the number of matching or homologous positions shared by the sequences.

"Cells," "host cells" or "recombinant host cells" are terms used interchangeably herein. It is understood that such terms refer not only to the particular subject cell but to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.

A "chimeric protein" or "fusion protein" is a fusion of a first amino acid sequence encoding one of the subject polypeptides with a second amino acid sequence defining a domain foreign to and not substantially homologous with any domain of the first polypeptide. A chimeric protein may present a foreign domain which is found (albeit in a different protein) in an organism which also expresses the first protein, or it may be an "interspecies", "intergenic", etc. fusion of protein structures expressed by different kinds of organisms.

4.3 Detailed Description of the Preferred Embodiments 4.3.1 Mytl Phosphorylation of Cdk2/CvcA

Xenopus Mytl kinase as originally isolated from Xenopus laevis was shown to phosphorylate Xenopus Cdc2/CycB on T14 and Y15 (Mueller et al. (1995) Science 270:86- 89). This reaction had an absolute requirement that Cdc2/CycB was CAK-phosphorylated on T161 as no phosphorylation could be detected using the T161A mutant. The reported reaction did not yield stoichiometrically labeled Cdc/CycB complex as radiolabeling using γ-³²P-ATP followed by phosphoamino acid analysis revealed that the ³²P was unequally distributed between phosphotyrosine (20%) and phosphothreonine (77%). Sub-stoichiometric phosphorylation reactions were also observed in the incomplete phosphorylation-induced mobility shift of Cdc2 as visualized by anti-Cdc2 antibodies in Western blotting experiments. No other Cdk/Cyclin (i.e. Cdk2/CycA, Cdk2/CycE, Cdk4/CycD) complexes were tested in this report.

The human analogue of Mytl was shown to phosphorylate human Cdc2/CycB on T14 and Y15 (Liu et al. (1997) Molec. CeL. Biol. 17:571-583; Booher et al. (1997) J. Biol. Chem. 272:22300-22306). Although not specifically addressed, this reaction appears to require CAK-phosphorylation as phosphorylation of the complex was usually evaluated by a loss in Cdc2 kinase activity. According to both reports, Mytl preferentially phosphorylates T14 over Y15, thereby not yielding quantitative phosphorylation on both residues. This is detectable in that the reaction with Mytl of the Cdc2-A14 mutant did not completely shut down the Cdc2 kinase activity, whereas Weel phosphorylation of this mutant did effectively inhibit the Cdc2 kinase activity. Also, as in the report using Xenopus Mytl, the lack of quantitative phosphorylation-induced mobility shifts of Cdc2 as visualized by anti-Cdc2 antibodies in Western blotting experiments was indicative of sub-stoichiometric phosphorylation.

Human Mytl reportedly does not phosphorylate human Cdk2/CycA or human Cdk2/CycE (Booher et al. (1997) J. Biol. Chem. 272:22300-22306). It will, however, phosphorylate human Cdc2/CycA, indicating that the Cdk subunit, not the cyclin subunit, is a key determinant in the recognition of a Cdk2/Cyclin complex by human Mytl.

Although some of the proteins involved in the cell cycle have been cloned by complementation in yeast, indicating that they can show species cross-reactivity, such species cross reactivity has not been specifically addressed in the inhibitory phosphorylation of Cdk/Cyclin complexes. Many kinases show a rather broad specificity for a consensus site which can be presented to the kinase in a variety of contexts, also as peptides. However, human Mytl appears to be a much more specific kinase. It recognizes its phosphorylation site on human Cdc2 (5-KIEKIGEGTYGVVYKGR-22), while not recognizing the equivalent site in human Cdk2 (5-KVEKIGEGTYGVVYKAR-22) (Booher et al. (1997) J. Biol. Chem. 272:22300-22306). Also, both the human and Xenopus Mytls appear to be specific for the subtly different conformation of the CAK-phosphorylated complex over the non-CAK phosphorylated complex (Mueller et al. (1995) Science 270:86-89; Russo et al. (1996) Nature Struct. Biol. 3:696-700). These results suggest that Mytl in general may not possess species cross-reactivity.

The first embodiment of this invention shows that Xenopus Mytl can be used to quantitatively phosphorylate human Cdk2/CycA complex, where the CycA is truncated to encompass residues 174-432, to generate Cdk2-pTpY/CycA. Given the literature precedence discussed above, this is surprising and novel because 1.) quantitative phosphorylation occurs on a 2.) Cdk2-containing complex using a 3.) cross-species kinase in the 4.) absence of CAK phosphorylation.

4.3.2 A Quantifiable Natural Substrate Assay for Cdc25

The preparation of reagent amounts of defined human Cdk2-pTpY/CycA allows the development of a quantifiable natural substrate assay of Cdc25. Recently, two publications have specifically addressed the production of native substrates for Cdc25. The first method relies on an "in vitro bathing protocol" (Clark & Gabrielli (1997) Anal. Biochem. 254:231- 235). GST-CycB is produced in insect cells and bound to GSH Separose beads. These are then bathed in asynchronous HeLa cell lysates where the following three sequential steps must occur without the benefit of enriched proteins or purified components: 1) Binding of GST- CycB to the Cdc2 kinase monomer; 2) CAK phosphorylation of the Cdc2/GST-CycB complex; 3) Weel and or Mytl phosphorylation of the CAK-phosphorylated complex. The complex-containing beads are then removed from the HeLa lysate, washed extensively, and removed from the beads by thrombin cleavage of the linker in the GST-CycB. Presumably the beads are a nonhomogenous and undefined mixture of the various intermediates of this preparation procedure. Also, undesired additional components such as pl3suc, p27, or p21 may have copurified using this protocol. No method is presented for characterization or purification of the final reagent. The assay procedure for this method of substrate preparation involves the indirect assay of Cdc2 kinase activation. Such an assay does not address the specific issue of Cdc25-catalyzed dephosphorylation of pT vs. pY in the natural substrate, given that the each of the species potentially generated by Cdc25 treatment (Cdc2- pTpY/CycB, Cdc2-pT/CycB, Cdc2-pY/CycB, Cdc2/CycB) presumably has a different specific kinase activity. Therefore, this assay procedure does not allow a true characterization of the interaction of Cdc25 with its natural substrate in a quantifiable manner.

The second recent report of a Cdc25 assay, although using some more refined techniques including a direct dephosphorylation assay, still relies on the use of an "in vitro bathing protocol" (Kumagai & Dunphy (1997) Methods Enzymol. 283:564-571). Xenopus His-CycB is produced in insect cells and bound to Ni-IDA beads. These are then bathed in a lysate of insect cells which overexpress Xenopus Cdc2. During this incubation, the Xenopus Cdc2/His-CycB complex forms and the complex is at least partially CAK phosphorylated (presumably by the native CAK from the insect cells). The complex-containing beads are then removed from the lysate, washed extensively, and removed from the beads by imidazole elution. The complex is then phosphorylated using purified Xenopus Mytl which can be done in the presence of labeled or unlabeled ATP. Once again, this procedure can lead to an undefined mixture of products, especially in regard to the phosphorylation state. Also, it again contains potentially undesired additional components such as pl3suc, p27, or p21. As a matter of fact, a variation of this procedure using Xenopus lysates instead of insect cell lysates and His-Cdc25 has been used to isolate novel Cdc25-binding proteins Kumagai & Dunphy (1996) Science 273:1377-1380). The direct assay procedure which is used to follow Cdc25 activity is performed using radiolabeled complex prepared using γ-³²P-ATP. Cdc25 treatment of this complex causes the reduction of label on Cdc2 which is detected following SDS-PAGE and autoradiography.

In summary, existing methods of natural substrate preparation and assay procedures for Cdc25 are lacking. The amounts of reagents which can be generated are low, nonhomogeneous in their state of phosphorylation and protein composition, and not quantifiable. The assay procedures are generally indirect and do not distinguish between dephosphorylation of pT and pY. Therefore, the second embodiment of this invention is the use of the Xenopus Mytl -phosphorylated human Cdk2-pTpY/CycA as a defined, quantifiable, and homogenous substrate for Cdc25. Cdk2-pTpY/CycA is a highly potent substrate for Cdc25 but not for other phosphatases. The preparation and use of Cdk2-pTpY/CycA in the manner of this invention allows the quantitation of the preference of phosphothreonine over phosphotyrosine in the context of the natural substrate, in contrast to peptidic substrates. 4.3.3 Detection of Unique Inhibitors of Cdc25

In many drug screening programs which test libraries of compounds and natural extracts, high throughput assays are desirable in order to maximize the number of compounds surveyed in a given period of time. Assays which are performed in cell-free systems, such as may be derived with purified or semi-purified proteins, are often prefeπed as "primary" screens in that they can be generated to permit rapid development and relatively easy detection of an alteration in a molecular target which is mediated by a test compound. Moreover, the effects of cellular toxicity and or bioavailability of the test compound can be generally ignored in the in vitro system, the assay instead being focused primarily on the effect of the drug on the molecular target, as may be manifest in an alteration of binding affinity between one of the subject proteins and other proteins with which they interact, in changes in enzymatic activity of one of the subject proteins, or in changes in a property of the molecular target manifest from binding to one of the regulatory proteins.

Due to the difficulties in preparing natural substrates for Cdc25, screening assays and kinetic/mechanism assays have relied on artificial substrates such as pNPP and mFP. There are several disadvantages to using these non-natural substrates. First, they react relatively poorly with Cdc25 compared to other phosphatases, this being especially true for pNPP where the K_m is >15 mM. Also, these substrates interact with the enzyme solely at the active site and not at other sites on the enzyme which may be used by the natural substrate. Therefore, their use may miss certain structural space in screening a compound library. For example, cyclin B has been proposed to form a part of the active site for Cdc25 in the dephosphorylation reaction of Cdc2 (Galaktionov & Beach (1991) Cell 67:1181-1194). The third embodiment of this invention takes advantage of the developed natural substrate assay to screen and characterize inhibitors of Cdc25, some of which may only be detected by a natural substrate assay. The sequential nature of the dephosphorylation reaction (i.e. pT before pY) permits identification of inhibitors of each of the dephosphorylation steps. These assays can be performed by a variety of techniques.

This invention includes within its scope assays wherein the ability of a test compound to disrupt and/or inhibit the dephosphorylation of one or both of the T14 and T15 residues. In one embodiment, the method comprises the steps of: (a) combining a compound to be assessed, the subject Cdc25 (purified or semipurified), and the phosphorylated Cdk/Cyc substrate; (b) maintaining the substrate/enzyme/test compound combination under conditions appropriate for the Cdc25 to dephosphorylate the native substrate complex; and (c) determining the extent to which the Cdc25 enzyme present in the combination dephosphorylated upon the substrate relative to a control, the control comprising the Cdc25 and the native substrate. If the subject rate of dephosphorylation of the native substrate is less than in the control, the compound is an inhibitor of the Cdc25 threonine or tyrosine phosphatase activity. The efficacy of the test compound can be assessed by generating dose response curves from data obtained using various concentrations of the test compound. Moreover, a control assay can also be performed to provide a baseline for comparison. Efficacy of an agent is based on producing a statistically significant change in the Cdc25 activity relative to the control. It will be understood that, in general, the order in which the reactants may be admixed can be varied, and can be admixed simultaneously.

4.3.3.a Detecting Inhibition of Dephosphorylation of Cdk/Cyc Complexes by Cdc25 A number of methods can be adapted from the art for measuring phosphatase- dependent dephosphorylation by Cdc25 of specific substrates, such as Cdk/Cyc complexes. For example, the sequential dephosphorylation by Cdc25 of the specific T14 and Y15 residues on Cdk can be measured by analyzing the release of inorganic phsophate from the phosphorylated complex. The inorganic phosphate can be most readily detected as radiolabeled phosphate (³²P or ³³P) when the phosphorylated Cdk2/CycA is prepared using γ- ³²P-ATP or γ-³³P-ATP. The inorganic phosphate which is released can be detected separately from the remaining radiolabeled substrate following TCA precipitation of the proteinaceous substrate and subsequent analysis of the soluble inorganic phosphate in the supernatant. The inorganic phosphate can also be detected by colorimetric techniques such as described, e.g., by Lanzaetta (1979). An enzymatic phosphate detection kit is also commercially available from New England Biolabs. To illustrate, dephosphorylation may be measured by (a) combining a compound to be assessed for its ability to inhibit dephosphorylation, the subject Cdc25 (purified or semipurified), and the bis-phosphorylated substrate of Cdc25; (b) maintaining the substrate/enzyme/test compound combination under conditions appropriate for the Cdc25 to act upon the substrate; and (c) determining at definite time intervals the amount of free inorganic phosphate released from the substrate compared to the control. The rate of release of phosphate, and thus the enzymatic activity of the Cdc25 phosphatase, can be determined from the slope of the graph resulting from a plot of inorganic phosphate released plotted on the ordinate versus time plotted on the abscissa.

Alternatively, the activity of Cdc25 can be followed by monitoring the loss of phosphate on the substrate. The loss of ³²P- or ³²P-labeled Cdk2/CycA complexes can be analyzed following isolation of the Cdk/Cyc complexes by general means such as SDS- PAGE, immunoprecipitation, gel filtration, acid precipitation, filter binding, etc.. The loss of phosphate on the Cdk/Cyc complex can also be detected by measuring the loss of the substrate's immunoreactivity against readily available anti-phosphothreonine and anti- phosphotyrosine antibodies. To illustrate, dephosphorylation may be measured by (a) combining a compound to be assessed for its ability to inhibit dephosphorylation, the subject Cdc25 (purified or semipurified), and the bis-phosphorylated substrate of Cdc25; (b) maintaining the substrate/enzyme/test compound combination under conditions appropriate for the Cdc25 to act upon the substrate; and (c) determining at definite time intervals the amount of the bis-phosphorylated Cdk/Cyc substrate remaining compared to the control.

In another embodiment, Cdc25 activity can be detected in a coupled assay which uses the intrinsic kinase activity of the Cdk/Cyc complex. In such assays, Cdc25 is contacted with a phosphorylated Cdk/Cyc complex under conditions wherein, absent an inhibitor of the Cdc25, the Cdc25 would dephosphorylate and activate the kinase activity of the Cdk/Cyc complex. Activation of the Cdk Cyc complex could be detected by conversion of a substrate for the activated kinase complex, such as phosphorylation of a histone HI protein using ³²P- labeled ATP, as is well described in the art.

4.3.3.b Detecting Disruption of Complex Formation between Cdk/Cyc and Cdc25

Additionally, this invention provides drug screening assays which are based upon the specificity of the natural substrates, to identify inhibitors which can bind to and/or disrupt binding between Cdc25 and a native Cdk/Cyc complex. Complex formation between the Cdc25 polypeptide and Cdk/Cyclin complex may be detected by a variety of techniques utilizing, for example, a catalytically inactive Cdc25 (i.e. active site Cys— >Ser mutant). Typically, one of the two components of interest will be immobilized to facilitate separation of complexed from uncomplexed forms as well as to accomodate automation fo the assay. In one embodiment, GST-fusion proteins (e.g. GST-Cdc25A) can be adsorbed onto glutathione Sepharose beads or glutathione-derivatized microtitre plates which are then combined with the appropriate Cdk/Cyc complex (e.g. Mytl -phosphorylated Cdk2/CycA). The modulation of the formation of complexes in the presence of a test compound can then be quantitated using detectably labeled proteins such as radiolabelled (e.g. ³²P, ³⁵S, ¹ C or ³H), fluorescently labeled (e.g. FITC), or enzymatically labeled polypeptides, by immunoassay, or by chromatographic detection or use of a biosensor based on, e.g., surface plasmon resonance.

4.3.4. Exemplary Uses of cdc25 Inhibitors

Based at least on the fact that Cdc25 is involved in the cell cycle, agents which modulate Cdc25 activity, identified according to the methods of the invention can be used to modulate cell growth, and/or differentiation and/or cell death. Thus, agents of the invention can be used as therapeutics to treat or prevent diseases in a subject which are characterized by an abnormal cell growth, differentiation, and/or cell survival, e.g., hyperproliferative or hypoproliferative diseases. Accordingly, the invention provides methods for treating or preventing a disease in a subject, comprising administering to the subject an effective amount of a pharmaceutical composition comprising a compound of the invention, such that the disease is treated or prevented in the subject. Hyperproliferative diseases that can be treated with compounds of the inventions include neoplastic and hyperplastic diseases, such as various forms of cancers and leukemias, and fibroproliferative disorders. Other hyperproliferative diseases that can be treated or prevented with the subject compounds include malignant conditions, premalignant conditions, and benign conditions. The condition to be treated or prevented can be a solid tumor, such as a tumor arising in an epithelial tissue. Accordingly, treatment of such a cancer could comprise administration to the subject compounds modulating the interaction of Cdc25 with its substrate(s). Other cancers that can be treated or prevented by administration of a compound of the invention include sarcomas and carcinomas, e.g., lung cancer, cancer of the colon, prostate, breast, ovary, esophagus, lung cancer, melanoma, seminoma, and squamous adenocarcinoma. Additional solid tumors within the scope of the invention include those that can be found in a medical textbook.

The condition to be treated or prevented can also be a soluble tumor, such as leukemia, either chronic or acute, including chronic or acute myelogenous leukemia, chronic or acute lymphocytic leukemia, promyelocytic leukemia, monocytic leukemia, myelomonocytic leukemia, and erythroleukemia. Yet other proliferative disorders that can be treated with a compound of the invention include heavy chain disease, multiple myeloma, lymphoma, e.g., Hodgkin's lymphoma and non-Hodgkin's lymphoma, and Waldenstroem's macroglobulemia.

Diseases or conditions characterized by a solid or soluble tumor can be treated by administrating a compound of the invention either locally or systemically, such that abeπant cell proliferation is inhibited or decreased. Methods for administering the compounds of the invention are further described below.

The invention also provides methods for preventing the formation and/or development of tumors. For example, the development of a tumor can be preceded by the presence of a specific lesion, such as a pre-neoplastic lesion, e.g., hyperplasia, metaplasia, and dysplasia, which can be detected, e.g., by cytologic methods. Such lesions can be found, e.g., in epithelial tissue. Thus, the invention provides a method for inhibiting progression of such a lesion into a neoplastic lesion, comprising administering to the subject having a preneoplastic lesion an amount of a compound of the invention sufficient to inhibit progression of the preneoplastic lesion into a neoplastic lesion.

In yet another embodiment, the invention provides a method for treating or preventing diseases or conditions characterized by abeπant cell differentiation. Accordingly, the invention provides methods for stimulating cellular differentiation in conditions characterized by an inhibition of normal cell differentiation which may or may not be accompanied by excessive proliferation. Alternatively, the compounds of the invention can be used to inhibit differentiation of specific cells. In another embodiment, the invention provides a method for enhancing the survival and/or stimulating proliferation and/or differentiation of cells and tissues in vitro . In a prefeπed embodiment, compounds of the inventions are used to promote tissue regeneration and/or repair (e.g., to treat nerve injury). For example, tissues from a subject can be obtained and grown in vitro in the presence of a compound of the invention, such that the tissue cells are stimulated to proliferate and/or differentiate. The tissue can then be readministered to a subject.

4.3.5 Pharmaceutical Preparations of cdc25 Inhibitors

Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining The Ld₅₀ (The Dose Lethal To 50% Of The Population) And The Ed₅o (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD₅o/ED₅₀. Compounds which exhibit large therapeutic induces are preferred. While compounds that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects.

The data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the method of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by high performance liquid chromatography.

Pharmaceutical compositions for use in accordance with the present invention may be formulated in conventional manner using one or more physiologically acceptable carriers or excipients. Thus, the compounds and their physiologically acceptable salts and solvates may be formulated for administration by, for example, injection, inhalation or insufflation (either through the mouth or the nose) or oral, buccal, parenteral or rectal administration. For such therapy, the compounds of the invention can be formulated for a variety of loads of administration, including systemic and topical or localized administration. Techniques and formulations generally may be found in Remmington's Pharmaceutical Sciences, Meade Publishing Co., Easton, PA. For systemic administration, injection is prefeπed, including intramuscular, intravenous, intraperitoneal, and subcutaneous. For injection, the compounds of the invention can be formulated in liquid solutions, preferably in physiologically compatible buffers such as Hank's solution or Ringer's solution. In addition, the compounds may be formulated in solid form and redissolved or suspended immediately prior to use. Lyophilized forms are also included.

For oral administration, the pharmaceutical compositions may take the form of, for example, tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g., pregelatinised maize starch, polyvinylpyπolidone or hydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystalline cellulose or calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc or silica); disintegrants (e.g., potato starch or sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulphate). The tablets may be coated by methods well known in the art. Liquid preparations for oral administration may take the form of, for example, solutions, syrups or suspensions, or they may be presented as a dry product for constitution with water or other suitable vehicle before use. Such liquid preparations may be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles (e.g., ationd oil, oily esters, ethyl alcohol or fractionated vegetable oils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates or sorbic acid). The preparations may also contain buffer salts, flavoring, coloring and sweetening agents as appropriate.

Preparations for oral administration may be suitably formulated to give controlled release of the active compound. For buccal administration the compositions may take the form of tablets or lozenges formulated in conventional manner. For administration by inhalation, the compounds for use according to the present invention are conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebuliser, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of e.g., gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch. The compounds may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.

The compounds may also be formulated in rectal compositions such as suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa butter or other glycerides.

In addition to the formulations described previously, the compounds may also be formulated as a depot preparation. Such long acting formulations may be administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, the compounds may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.

Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration bile salts and fusidic acid derivatives. In addition, detergents may be used to facilitate permeation. Transmucosal administration may be through nasal sprays or using suppositories. For topical administration, the compounds of the invention are formulated into ointments, salves, gels, or creams as generally known in the art. A wash solution can be used locally to treat an injury or inflammation to accelerate healing.

Other features and advantages of the invention will be apparent from the following detailed description, and from the claims. The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature. See, for example, Molecular Cloning A Laboratory Manual, 2nd Ed., ed. by Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory Press: 1989); DNA Cloning, Volumes I and II (D. N. Glover ed., 1985); Oligonucleotide Synthesis (M. J. Gait ed., 1984); Mullis et al. U.S. Patent No. 4,683,195; Nucleic Acid Hybridization (B. D. Hames & S. J. Higgins eds. 1984); Transcription And Translation (B. D. Hames & S. J. Higgins eds. 1984); Culture Of Animal Cells (R. I. Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells And Enzymes (IRL Press, 1986); B. Perbal, A Practical Guide To Molecular Cloning (1984); Methods In Enzymology (Academic Press, Inc., N.Y.); Gene Transfer Vectors For Mammalian Cells (J. H. Miller and M. P. Calos eds., 1987, Cold Spring Harbor Laboratory); Methods In Enzymology, Vols. 154 and 155 (Wu et al. eds.), Immunochemical Methods In Cell And Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987); Handbook Of Experimental Immunology, Volumes I- IV (D. M. Weir and C. C. Blackwell, eds., 1986).

5. Exemplification

The invention now being generally described will be more readily understood by reference to the following examples which are included merely for purposes of illustration of certain aspects and embodiments of the present invention, and are not intended to limit the invention.

5.1 Phosphorylation of human Cdk2/CycA by Xenopus Mytl 5.1.1 Protein Reagents

Mytl from Xenopus laevis was cloned as a GST-fusion protein and prepared as follows: A 1.8 Kb fragment containing full length myt-1 c-DNA was amplified from lambda phage DNA prepared from a Xenopus laevis oocyte c-DNA library (Clonetech) using primers of the sequence (amino terminal) GGCCCGGGATGCCTGTTCCAGGGG and (carboxy terminal) GGCCCGGGGTCATGGCGATATCATGAA. The myt-1 cDNA was ligated into the PCRII cloning vector to generate PCRIImyt-1, and sequenced. PCRIImyt-1 was then digested with Smal and the myt-1 cDNA was ligated into Smal-cut pACG2T. The resulting plasmid was transfected into insect cells using the Baculogold transfection system. Expression was optimized by plaque purification of the recombinant baculovirus. The frozen cells from a 4 L infection were thawed in 50 mL lysis buffer (10 mM HEPES (pH 7.4), 150 mM NaCl, 5 mM EGTA, 0.5% Triton-XlOO and the protease inhibitors PIN, TPCK, TLCK at 0.1 mg/mL) and subjected to mild sonication. GST-Mytl was batch-bound to 5 mL of GSH- Sepharose. Following washes with 20 volumes of lysis buffer containing 1 M NaCl, the column was washed with 20 volumes of Myt-kinase buffer (50 mM Tris-HCl (pH 7.5), 100 mM NaCl, 10 mM MgCl2, 1 mM DTT, and 0.1% Triton X-100). The protein was stored in kinase buffer at -70°C bound to the GSH-beads.

Weel from Xenopus laevis was cloned as a GST-fusion protein and prepared as described previously (Parker & Piwnica-Worms (1992) Science 257:1955-1957). GST-Weel was stored in Wee-kinase buffer (40 mM HEPES (pH 7.5), 50 mM NaCl, 10 mM MgCl₂, 10 mM MnCl₂, 1 mM DTT) at -70°C bound to the GSH-beads.

The human Cdk2/CycA complex, where the CycA is truncated to encompass residues 174-432, was prepared as described previously (Jeffrey et al. (1995) Nature 376:313-320). CAK-phosphorylated human Cdk2/CycA complex was prepared as described previously (Russo et al. (1996) Nature Struct. Biol. 3:696-700).

5.1.2 Mytl Reaction and Work-Up Conditions

Reaction conditions to achieve quantitative phosphorylation of Cdk2/CycA on T14 and Y15 were established by testing various ATP concentrations, kinase to substrate ratios, and reaction times. All Mytl phosphorylations were performed in Myt-kinase buffer. It was found that the reaction is dependent on the concentration of of ATP, with > 2.5 mM needed to achieve quantitative Mytl phosphorylation. Systematic variation of the ratio of kinase to Cdk2/CycA showed that 0.25 to 0.5 equivalents of GST-Mytl were required per equivalent of the substrate to achieve quantitative phosphorylation. Time-courses run to evaluate the extent of phosphorylation showed 2.5 h to be sufficient, with shorter reaction times yielding sub- stoichiometric phosphorylation and longer reaction times leading to a reduction in the yield of recoverable Cdk2/CycA protein. Under conditions of radiolabeling, 3.3 mM of γ-³²P-ATP at 200 Ci/mol was used. All reactions were terminated by removal of the kinase-containing beads by centrifugation at 1500 x g for 3 min..

Two different methods were used in the work-up of the phosphorylation reaction in order to remove unincorporated ATP and the kinase reaction buffer components. The first method used G-50 sizing chromatography in 40 mM HEPES (pH 7.5), 200 mM NaCl, and 5 mM DTT. The column size was 0.7 x 20 cm and the sample volume loaded was 900 μL. The protein-containing fractions was collected and pooled. The second method used POROS-HS chromatography. The reaction solution was diluted to 30 mM NaCl final concentration using 10 mM HEPES (pH 7.5). The protein was then bound to a 1 mL column of POROS-HS and washed extensively with 40 mM HEPES, 30 mM NaCl, and 5 mM DTT. The phosphorylated complex was then eluted using this buffer containing an additional 370 mM NaCl.

Phosphorylation reactions using Weel and their work-up were performed in an analogous manner with the following differences. No detergent was used in the phosphorylation reaction, which contained 300 μM ATP instead of the 3.3 mM used in the Mytl reaction.

5.1.3 Characterization of the Phosphorylated Complex Cdk2-pTpY/CycA

Phosphorylation of Cdk2/CycA on T14 and Y15 was monitored and quantitated by radiolabeling, mass spectrometry, and phosphoamino acid analysis. A reaction containing 1 μg GST-Mytl and 3.3 mM γ-³²P-ATP in the presence (Fig. 1.1, lane 1) or absence (Fig 1.1, lane 2) of 3 μg of Cdk2/CycA was performed as described above and was quenched with Laemmli buffer prior to removal of the GST-Mytl beads. The reaction mixtures were boiled for 3 min and then subjected to SDS-PAGE and autoradiography. As indicated by the aπows, Mytl undergoes self -phosphorylation as well as phosphorylating Cdk2. Quantitation of the amount of phosphorylation indicates the incorporation of 0.5 to 2 equivalents of phosphate per Cdk2/CycA complex.

Direct and strongly corroborating evidence for the incorporation of two phosphate moieties into Cdk2 was obtained by LC-MS. Following Mytl phosphorylation (using unlabeled ATP) and G-50 chromatography, the phosphorylated Cdk2/CycA samples were subjected to reverse phase liquid chromatography in 0.1% TFA and eluted using an acetonitrile gradient in 0.1% TFA. The eluted Cdk2 was subjected to direct inject electrospray mass spectrometry. Fig. 1.2 shows the conversion of Cdk2 (expected MW = 33,975 Da; MW determined by MS = 33,971 Da) to Cdk-pTpY (expected MW = 34,135 Da; MW determined by MS = 34,130 Da). The observed increase in the molecular weight of 160 Da (= 2 x 80 Da) is consistent with the incorporation of two phosphates per Cdk2 monomer. The molecular weight of CycA remained unchanged by Myt treatment (not shown).

In order to provide evidence that Mytl was phosphorylating both T14 and Y15 residues, as expected from the literature precedent, the Mytl -phosphorylated complex (using γ-³²P-ATP) was boiled in HCl and was subjected to phosphoamino acid analysis as described by (Cooper et al. (1983) Methods Enzymol 99:387-402). Fig. 1.3 shows the autoradiograph which indicates the co-migration of ³²P with phosphothreonine and phosphotyrosine, but not phosphoserine, standards as detected by Ninhydrin spray.

In order to provide additional evidence that Mytl phosphorylation was directed at T14 and Y15, Cdk2-pTpY was subjected to a dephosphorylation using GST-Cdc25A or the catalytically inactive mutant GST-Cdc25A (C430S). Treatment of 3 μg of Cdk2-pTpY/CycA prepared using γ-³²P-ATP, with GST-Cdc25A (Fig. 1.4, lane 2), but not with the C430S mutant (Fig. ID, lane 3) led to complete dephosphorylation of the complex, again consistent with Myt-1 phosphorylation of being directed at T14 and Y15.

5.1.4 Xenopus Myt phosphorylation of CAK-phosphorylated human Cdk2/CycA

CAK phosphorylation of T160 on Cdk2 leads to an downward shift in the migration of Cdk2 as detected by SDS-PAGE (Gu et al. (1992) EMBO J. 11 :3995-4005). One can therefore easily analyze the relative Xenopus Mytl phosphorylation levels of CAK- vs. non- CAK-phosphorylated Cdk2/CycA. A small scale phosphorylation reaction containing equal amounts of either CAK-phosphorylated human Cdk2/CycA or unphosphorylated human Cdk2/CycA was performed using Xenopus Myt-1. Qualitative analysis by autoradiography following gel electrophoresis of the reaction mixture containing γ-³²P-ATP indicated equal phosphorylation yields for the two different Cdk2/CycA species.

Briefly, autoradiography following SDS-PAGE of a reaction mixture containing 1 μg Xenopus GST-Mytl and 3.3 mM γ-³²P-ATP in the presence of 3 μg of unphosphorylated Cdk2/CycA (lane 1) or 3 μg of CAK-phosphorylated Cdk2/CycA (lane 2) demonstrating that Mytl phosphorylates both the non-CAK- and CAK-phosphorylated complexes with approximately equal efficiency. Note the slightly slower migrating CAK-phosphorylated form of Cdk2 compared to the non-CAK-phosphorylated form.

5.2 Phosphothreonine Hydrolysis from Cdk2-pTpY Can Be Used To Assay Cdc25 5.2.1 Cdk2-pTpY is a highly reactive and specific substrate toward Cdc25

Cdc25A and Cdc25C were prepared as GST fusion proteins as described previously (Galaktionov & Beach (1991) Cell 67:1181-1194). Cdc25B(392-580), the catalytic domain of Cdc25B, was prepared as described (Gottlin et al. (1996) I. Biol. Chem. 271:27445-27449) and Cdc25A(324-524), Cdc25A(337-524), Cdc25A(337-504) and Cdc25C(279-473), catalytic domains of Cdc25A and Cdc25C were prepared in an analogous manner. VHR was purified from an E. coli overexpression strain to 90% homogeneity as described (Denu et al. (1995) J. Biol. Chem. 270:3796-3803). PTPlb was purified from an E. coli overexpression strain to 95% homogeneity as described (Tonks et al. (1988) J. Biol. Chem. 263:6731-6737).

All phosphatase reactions were performed in 50 mM Tris-HCl (pH 8.0), 50 mM NaCl, 1 mM ΕDTA, 1 mM DTT at 25°C. Reactions using p-nitrophenyl phosphate (pNPP, Sigma) were followed by continuous UV-Vis spectroscopy at 410 nm (e = 18,000 M^cnr¹). Phosphopeptide reactions were either followed by continuous monitoring of the increase of absorbance at 295 nm (e = 280 M^cπr¹) or by HPLC analysis of fixed time points (reverse phase liquid chromatography eluted using an acetonitrile gradient in 0.1% TFA). Reactions with the natural substrate were performed in the presence of 1 mg/mL bovine serum albumin using Cdk2-pTpY prepared using γ-³ P-ATP as described above. Fixed time-points were quenched by the addition of 0.3 equivalents (by volume) of 30% TCA. The supernatant containing the released phosphate was subjected to scintillation counting following centrifugation of the precipitated protein at 14K and 4°C for 10 min..

The time course of a typical biphasic dephosphorylation reaction of Cdk2-pTpY/CycA catalyzed by Cdc25A is shown in Fig. 2.1. The first important result to note is the fast reaction rate. Fits to the initial linear part of the reaction indicate a k^/Km of 250,000 M^{_ 1}s^{_ 1}

(see Table 1). This is 15,000-fold higher than the best peptidic substrate investigated for purposes of comparison and 20-fold higher than the best artificial substrate, mFP. The K_m for the complex was estimated to be around 1 uM. Thus, Cdc25A is indeed a highly potent phosphatase. This assay allows the determination of exact rate constants because the concentration of the substrate is defined and known. Additionally, this result provides strong quantitative support for Cdc25A and Cdk2/CycA functioning as a natural enzyme/substrate pair in the control of the cell cycle. Some preliminary evidence for the selectivity of Cdc25 derived from Drosophila toward Cdc2/CycB substrates derived from Xenopus (over random phosphotyrosine-containing peptides) has been reported, but this cannot be considered a physiologically relevant reaction and no true quantitation was undertaken (Dunphy & Kumagai (1991) Cell 67:189-196).

By investigating the reactivity of Cdk2-pTpY/CycA toward other phosphatases, the high specificity of Cdc25 immediately becomes apparent. As seen in Figure 2.2, Cdc25A was by far the best phosphatase. Cdc25C was a very poor phosphatase for Cdk2-pTpY with approximately 100-fold lower activity. Interestingly, the highly reactive and significantly more promiscuous protein phosphatases VHR and PTPlb (Denu et al. (1995) J. Biol. Chem. 270:3796-3803; Tonks et al. (1988) J. Biol. Chem. 263:6731-6737) showed very little reactivity towards Cdk2-pTpY/CycA. Such lack of reactivity towards Cdk2-pTpY/CycA is noteworthy given that these phosphatases have been shown to react efficiently with a variety of non-specific peptidic substrates.

The reactivity towards Mytl -phosphorylated Cdk Cyc substrates can be used to define active catalytic domains compared to inactive domains which may be useful for assay development or structural biology. For example, the catalytic domain Cdc25A(324-524) is just as active as the full-length protein (Table 2). The slightly shorter catalytic domain, Cdc25A(337-524), although fully active against pNPP and mFP substrates, is about 10-fold less potent when measured by its activity against Cdk2-pTpY/CycA. This result is coπoborated by an in vivo measure of Cdc25 activity, namely the oocyte activation assay performed by the method of (Rime et al. (1994) Biol. Cell 82: 11-22) (Figure 2.3). An even shorter catalytic domain, Cdc25A(337-504), is only slightly reduced in its activity toward the artificial substrates pNPP and mFP, yet is completely inactive against the natural substrate as tested in the assay against Cdk2-pTpY/CycA and the oocyte activation assay (Table 2 and Figure 2.3). Thus, Cdc25A(324-524) is a more relevant target than the slightly shorter catalytic domain constructs for use in screening assays, and it is a more relevant domain to pursue in structural biology efforts.

Table 2: S ecificity of Varying Cdc25A Constructs toward Cdk2- T Y/CycA

5.2.2 Phosphothreonine is the Preferred Substrate

Analyzing the reaction time-course of Cdc25 with Xenopus Myt-phosphorylated human Cdk2/CycA in detail reveals the important observation that phosphothreonine is the preferπed target of Cdc25 in the context of the natural substrate. The Cdc25-catalyzed dephosphorylation of Cdk2-pTpY exhibits a biphasic nature when one examines the complete reaction time-course (Figure 2.1, insert). The presence or addition of reaction product, i.e. unphosphorylated complex, does not affect the dephosphorylation rates, discounting the possibility that product inhibition is the source of the biphasic kinetics. Therefore, this biphasic behavior is most easily explained by the presence of two substrates which are dephosphorylated at different rates. Phosphoamino acid analysis shows that both phosphothreonine and phosphotyrosine are present at the early time points, whereas only phosphotyrosine is detected at the time points taken near 50% completion of the reaction (Fig. 2.4). This shows that phosphothreonine is the prefeπed initial target of Cdc25A, leading to a mono-phosphorylated Cdk2-pY/CycA intermediate which is subsequently dephosphorylated at a slower rate. The rate constants from the linear portions of each of these reactions are 9- fold different. These results stand in stark contrast to the observed preference for phosphotyrosine residues in phosphopeptide substrates (see Table 1). Although a preference for phosphothreonine in the natural substrate reaction of Cdc25 (from starfish oocytes) has been observed previously Borgne & Meijer (1996) I. Biol. Chem. 271:27847-27854), this is the first example of a quantitative analysis of this reaction where the assay can be used to screen or characterize inhibitors of the Cdc25-catalytzed reaction (see below).

The preference for phosphothreonine over phosphotyrosine in Mytl -phosphorylated Cdk2/CycA was confirmed by performing dephosphorylation reactions of Xenopus Weel- phosphorylated Cdk2/CycA which generates only Cdk2-pY/CycA. In these reactions, the rate of dephosphorylation matched the slower rate of dephosphorylation observed using Xenopus Mytl-phosphorylated Cdk2-pTpY/CycA (Table 1). 5.3. Inhibitor Screens

IC-50 determinations were performed under the phosphatase reaction conditions described above by adding serial dilutions of inhibitor to separate reactions vessels and quantitating the amount of Cdc25 activity. Cdc25 activity was detected either by measuring mFP hydrolysis or ³²P-release from radiolabeled Cdk2-pTpY/CycA, as described above. Two different classes of inhibitors are presented below in order to exemplify the power and the advantages of the natural substrate assay.

As seen in Fig. 3.1, tungstate is an inhibitor with similar potency when assayed against either mFP or Cdk2-pTpY/CycA. This is not surprising, given that the assay conditions are similar in both experiments. In experiments where the substrate concentrations of either mFP or Cdk2-pTpY/CycA are varied, no significant changes are seen in the IC-50 values (data not shown), which then allows the calculation of a true Kj for tungstate of 78 ± 14 μM or 74 ± 9 μM, vs. mFP or Cdk2-pTpY/CycA, respectively.

As seen in Fig. 3.2, p27(Kipl) is a potent inhibitor of Cdc25 activity (Ic-50 = 78 nM), but only when measured against the natural substrate assay. When using mFP, it shows no significant effect on the reaction. This is an example, therefore, of an inhibitor of Cdc25 activity which would not be detected in an assay using an artificial substrate.

6. Equivalents

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, numerous equivalents to the specific assay and reagents described herein. Such equivalents are considered to be within the scope of this invention and are covered by the following claims.

Claims

Claims

1. A method of identifying a compound which is an inhibitor of human Cdc25 phosphatase activity, comprising the steps of: a) combining, in a reaction mixture, a Cdc25 phosphatase, phosphorylated complexes of mammalian Cdk and cyclin polypeptides (Cdk/cyclin complex), and a test compound, wherein (i) the Cdc25 phosphatase will dephosphorylate the phosphorylated Cdk/cyclin complex in the absence of an inhibitor of the Cdc25 phosphatase, and (ii) the reaction mixture is defined with respect to phosphorylation of the Cdk/cyclin complex, or is substantially free of Cdk/cyclin associated proteins, or both; and b) detecting T14 and/or Y15 dephosphorylation of the phosphorylated Cdk/cyclin complex, wherein the test compound is an inhibitor of the Cdc25 phosphatase if it decreases the ability of the Cdc25 phosphatase to dephosphorylate the phosphorylated Cdk/cyclin complex.

2. The method of claim 1, wherein dephosphorylation of the phosphorylated Cdk/cyclin complex is determined by detecting the release of free inorganic phosphate (³¹P,³²P or 3³P).

3. The method of claim 1, wherein the Cdk/cyclin complex is composed of human Cdk and cyclin polypeptides.

4. The method of claim 1, wherein the Cdk/cyclin complex is a Cdk2/CycA complex.

5. The method of claims 1-4, wherein Thr-161 of the Cdk protein in the Cdk/cyclin complex is phosphorylated.

6. The method of claims 1-4, wherein the Cdk is phosphorylated by a Mytl kinase.

7. The method of claims 1-4, wherein the Mytl kinase is derived from Xenopus, starfish, yeast, or Drosophila.

8. The method of claims 1-4, wherein the Cdk is phosphorylated by a Weel kinase. from Xenopus is used to phosphorylate the Cdk2/CycA complex.

. The method of claim 8, wherein the Weel kinase is derived Xenopus, starfish, yeast, or Drosophila.

10. The method of claims 1 or 3, wherein the Cdk/cyclin complex includes less than 25 percent (w/w protein) of pl3^sucl, INK4 or WAFl/Cipl proteins.

11. The method of claim 1 , wherein the Cdk/cyclin complex is comprised of truncated or fusion protein forms of Cdk and cyclin proteins.

12. The method of claim 1, wherein the Cdk/cyclin complex is selected from the group consisting of Cdc2/CycB, Cdk2/CycE, Cdk4/CycD, and Cdk6/CycD or any fragments thereof.

13. The method of claim 1, wherein the test compound is a peptide, a nucleic acid, a carbohydrate, a small organic molecule, or natural product extract or fraction thereof.

14. The method of claim 1, further comprising a step of formulating a pharmaceutical composition comprising one or more of the compounds, or derivatives thereof, identified as inhibitors of cdc25 phosphatase.

15. A method of identifying a compound which is an inhibitor of human Cdc25 phosphatase activity, comprising the steps of: a) combining, in a reaction mixture, a Cdc25 phosphatase, phosphorylated complexes of mammalian Cdk and cyclin polypeptides (Cdk/cyclin complex), and a test compound, wherein (i) the Cdc25 phosphatase bind to the phosphorylated Cdk/cyclin complex in the absence of an inhibitor of the Cdc25 phosphatase, and (ii) the reaction mixture is defined with respect to phosphorylation of the Cdk/cyclin complex, or is substantially free of Cdk/cyclin associated proteins, or both; and b) detecting the formation of a complex between the Cdk/cyclin complex and the Cdc25 phosphatase, wherein the test compound is an inhibitor of the Cdc25 phosphatase if it decrease the ability of the Cdc25 phosphatase to bind the phosphorylated Cdk/cyclin complex.

16. The method of claim 15, wherein the Cdc25 phoshpatase is a catalytically inactive mutant which retains the ability to bind to the phosphorylated Cdk/cyclin complex.

17. A composition comprising a Cdk/cyclin complex, the composition being homogenous with respect to the sequence of the cdk and cyclin proteins, having a defined phosphorylation state, and substantially lacking other proteins which bind the cdk, the cyclin or the Cdk/cyclin complex.

18. The composition of claim 17, wherein at least 10% of the Cdk/cyclin complex are phosphorylated complexes.

19. The composition of claim 17 or 18, wherein at least 75% of the phosphorylated Cdk/cyclin complexes are bis-phosphorylated.

20. The composition of claims 17, wherein the cdk and cyclin are mammalian cdk and cyclin proteins or fragments thereof.

21. A composition of 20, wherein the cdk and cyclin are human cdk and cyclin proteins or fragments thereof.

22. A composition of 17 or 20, wherein the cdk is selected from the group consisting of cdc2, cdk2, cdk4, and cdk6 and wherein the cyclin is selected from the group consisting of cyclin A, cyclin B, cyclin C, cyclin D and cyclin E.

23. A composition of 22, wherein the complexes is selected from the group consisting of cdk2/ cyclin A, Cdk4/cyclin A, cdk6/cyclin A, cdk2/ cyclin A, cdk2/ cyclin B, cdk2/ cyclin E, and cdk2/ cyclin B complexes.

23. The composition of claim 17, wherein Thr-161 of the Cdk is phosphorylated.