CA2163524C - Assay and reagents for identifying anti-proliferative agents - Google Patents

Abstract

The present invention makes available assays and reagents for identifying antiproliferative agents, such as mitotic and meiotic inhibitors. The present assay provides a simple and rapid screening test which relies on scoring for positive cellular proliferation as indicative of anti-mitotic or anti-meiotic activity, and comprises contacting a candidate agent with a cell which has an impaired cell-cycle checkpoint and measuring the level of proliferation in the presence and absence of the agent. The checkpoint impairment is such that it either causes premature progression of the cell through at least a portion of a cell-cycle or inhibition of normal progression of the cell through at least a portion of a cell-cycle, but can be off- set by the action of an agent which inhibits at least one regulatory protein of the cell cycle (e.g., cdc25) in a manner which counter- balances the effect of the impairment.

Description

Assay and Reagents for Identifying Anti proliferative Agents Background of the Invention Entry of cells into mitosis characteristically involves coordinated and simultaneous events, which include, for example, cytoskeletal rearrangements, disassembly of the nuclear envelope and of the nucleoli, and condensation of chromatin into chromosomes.
Cell-cycle events are thought to be regulated by a series of interdependent biochemical steps, with the initiation of late events requiring the successful completion of those proceeding them. In eukaryotic cells mitosis does not normally take place until the G1, S and G2 phases of the cell-cycle are completed. For instance, at least two stages in the cell cycle are regulated in response to DNA damage, the G 1 /S and the G2/M transitions. These transitions serve as checkpoints to which cells delay cell-cycle progress to allow repair of damage before entering either S phase, when damage would be perpetuated, or M phase, when breaks would result in loss of genomic material. Both the G1/S and G2/M checkpoints are known to be under genetic control as there are mutants that abolish arrest or delay which ordinarily occur in wild-type cells in response to DNA damage.
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 appropriate time. These regulatory proteins can provide exquisitely sensitive feedback-controlled circuits that can, for example, prevent exit of the cell from S
phase when a fraction of a percent of genomic DNA remains unreplicated (Dasso et al. (1990) Cell 61:811-823) and can block advance into anaphase in mitosis until all chromosomes are aligned on the metaphase plate (Rieder et al. (1990) J. Cell Biol. 110:81-95). In particular, the execution of various stages of the cell-cycle is generally believed to be under the control of a large number of mutually antagonistic kinases and phosphatases. For example, genetic, biochemical and morphological evidence implicate the cdc2 kinase as the enzyme responsible for triggering 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). The similarities between the checkpoints in mammalian cells and yeast have suggested similar roles for cdc protein kinases across species. In support of this hypothesis, a human cdc2 gene has been found that is able to substitute for the activity of an S. Pombe cdc2 gene in both its G 1/S and G2/M roles (Lee et al (1987) Nature 327:31). Likewise, the fact that the cdc2 homolog of S.
Cerevisae (cdc28) can be replaced by the human cdc2 also emphasizes the extent to which the basic cell-cycle machinery has been conserved in evolution.
As mitosis progresses, the cdc2 kinase appears to trigger a cascade of downstream _2_ 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 phosphorylating a wide variety of mitotic substrates such as nuclear lamins, histones, and microtubule-associated proteins (Moreno et al. (1990) Cell 61:549-551; and Nigg ( 1991 ) Semin. Cell Biol. 2:261-270). The cytoskeleton of cultured cells entering mitosis is rearranged dramatically. Caldesmon, an actin-associated protein, has also been shown to be a cdc2 kinase substrate (Yamashiro et al. ( 1991 ) Nature 349:169-172), and its phosphorylation may be involved in induction of M-phase-specific dissolution of actin cables. The interphase microtubule network disassembles, and it replaced by a mitosis-specific astral array emanating from centrosomes. This rearrangement has been correlated with the presence of mitosis-specific cdc2 kinase activity in cell extracts (Verde et al (1990) Nature 343:233-238). Changes in nuclear structure during mitotic entry are also correlated with cdc2 kinase activity. Chromatin condensation into chromosomes is accompanied by cdc2 kinase-induced phosphorylation of histone H I (Langan et al. ( 1989) Molec. Cell. Biol. 9:3860-3868), nuclear envelope dissolution is accompanied by cdc2-specific phosphorylation of lamin B (Peter et al. (1990) Cell 61:591-602) nucleolar disappearance is coordinated with the cdc2-dependent phosphorylation of nucleolin and N038.
The activation of edc2 kinase activity occurs during the M phase and is an intricately regulated process involving the concerted binding of an essential regulatory subunit (i.e., a cyclin) and phosphorylation at multiple, highly conserved positions (for review, see Fleig and Gould (1991) Semin. Cell Biol. 2:195-204). The complexity of this activation process most likely stems from the fact that, as set out above, the initiation of mitosis must be keyed into a number of signal transduction processes whose function is to guard against the inappropriate progression of the cell-cycle. In particular, the cell employs such signaling mechanisms to guarantee that mitosis and cytokinesis do not occur unless cellular growth and genome duplication have occurred in an accurate and timely manner.
The cdc2 kinase is subject to multiple levels of control. One well-characterized mechanism regulating the activity of cdc2 involves the phosphorylation of tyrosine, threonine, and serine residues; the phosphorylation level of which varies during the cell-cycle (Draetta et al. (1988) Nature 336:738-744; Dunphy et al. (1989) Cell 58:181-191; Morla et al.
(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 Tyr-15 and Thr-14, two residues located in the putative ATP binding site of the kinase, negatively regulates kinase activity.

21 fi 3 y ~:~.
This inhibitory phosphorylation of cdc2 is mediated at least impart by the weel and mild tyrosine kinases (Russet et al. ( 1987) Cell 49:559-567; Lundgren et al. ( 1991 ) Cell 64:1111-1122; Featherstone et al. ( 1991 ) Nature 349:808-811; and Parker et al. ( 1992) PNAS 89:2917-2921 ). 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, dephosphorylation of the cdc2-inactivating Thr-14 and Tyr-15 residues occurs leading to activation of the cd: .~' 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). Recent evidence indicates that both the cdc25 phosphatase and the cdc2-specific tyrosine kinases are detectably active during interphase, suggesting that there is an ongoing competition between these two activities prior to mitosis (Kumagai et al. (1992) Cell 70:139-151; Smythe et al. (1992) Cell 68:787-797; and Solomon et al. (1990) Cell 63:1013-1024. This situation implies that the initial decision to enter mitosis involves a modulation of the equilibrium of the phosphorylation state of cdc2 which is likely controlled by variation of the rate of tyrosine dephosphorylation of cdc2 and/or a decrease in the rate of its tyrosine phosphorylation. A variety of genetic and biochemical data appear to favor a decrease in cdc2-specific tyrosine kinase activity near the initiation of mitosis which can serve as a triggering step to tip the balance in favor of cdc2 dephosphorylation (Smythe et al. ( 1992) Cell 68:787-797; Matsumoto et al. ( 1991 ) Cell 66:347-360; Kumagai et al. (1992) Cell 70:139-151; Rowley et al. (1992) Nature 356:353-355; and Enoch et al. (1992) Genes Dev. 6:2035-2046). Moreover, recent data suggests that the activated cdc2 kinase is responsible for phosphorylating and activating cdc25. This event would provide a self amplifying loop and trigger a rapid increase in the activity of the cdc25 protein, ensuring that the tyrosine dephosphorylation of cdc2 proceeds rapidly to completion (Hoffmann et al. (1993) EMBOJ 12:53).

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Summary of the Invention The present invention makes available assays and reagents for identifying anti-proliferative agents, such as mitotic and meiotic inhibitors. The present assay provides a simple and rapid screening test which relies on scoring for positive cellular proliferation as indicative of anti-mitotic or anti-meiotic activity, and comprises contacting a candidate agent with a cell which has an impaired cell-cycle checkpoint and measuring the level of proliferation in the presence and absence of the agent. The checkpoint impairment is such that it either causes premature progression of the cell through at least a portion of a cell-cycle or inhibition of normal progression of the cell through at least a portion of a cell-cycle, but can be off set by the action of an agent which inhibits at least one regulatory protein of the cell-cycle in a manner which counter-balances the effect of the impairment. In one embodiment of the assay, anti-mitotic agents can be identified through their ability to rescue an otherwise hyper-mitotic cell from mitotic catastrophe (e.g. cell death) by inhibiting the activity of at least one regulatory protein of the cell-cycle which acts as a mitotic activator.
In another embodiment of the assay, an anti-mitotic agent can be identified by its ability to induce mitosis in an otherwise hypo-mitotic cell by inhibiting the activity of at least one regulatory protein of the cell-cycle which acts as a negative regulator of mitosis. In yet another embodiment of the invention, anti-meiotic agents can be identified by their ability to bring about faithful meiosis of an otherwise hyper-meiotic or hypo-meiotic cell.
The impaired checkpoint can be generated, for example, by molecular biological, genetic, and/or biochemical means. The checkpoint to be impaired can comprise a regulatory protein or proteins which control progression through the cell-cycle, such as those which control the G2/M transition or the G1/S transition. By way of example, the impaired checkpoint can comprise regulatory proteins which control the phosphorylation/
dephosphorylation of a cdc protein kinase, such as the gene products of weel, mild, or niml.
The cell used in the assay (reagent cell) can be generated so as to favor scoring for anti-proliferative agents which specifically inhibit a particular cell-cycle activity. For example, if it is desirable to produce an inhibitor to a cdc25 phosphatase activity, a hyper-mitotic or hyper-meiotic cell can be generated which would be rescued from mitotic or meiotic catastrophe by partial inhibition of cdc25.
Furthermore, the hyper- and hypo-proliferative cells of the present assay, whether for identifying anti-mitotic or anti-meiotic agents, can be generated so as to comprise heterologous cell-cycle proteins (i.e. cross-species expression). For example, a cdc25 homolog from one species can be expressed in the cells of another species where it has been shown to be able to rescue loss-of function mutations in that host cell. For example, ~a hyper mitotic Schizosaccharomyces cell, such as Schizosaccharomyces pombe, can be constructed so as to comprise an exogenous cdc25 phosphatase and a conditionally impairable weel protein kinase. The exogenous cdc25 can be, for example, a human cdc25 homolog, or alternatively, a cdc25 homolog from a human pathogen.
Description of the Drawings Figure 1 is a schematic representation of the construction of the "5'-half ura4-adh promoter- cdc25A-3'-half ura4" nucleic acid fragment of Example 1 for transforming ura4+
S. pombe cells.
Figure 2 is a schematic representation of the construction of the "5'-half ura4-adh promoter- cdc25B-3'-half ura4" nucleic acid fragment of Example 2 for transforming ura4+ S.
pombe cells.
Figure 3 is a schematic representation of the construction of the pART3-cdc25C
plasmid of Example 3.
Figure 4 is a schematic representation of the construction of the "5'-half ura4-adh promoter- cdc25C-3'-half ura4" nucleic acid fragment of Example 3 for transforming ura4+ S.
pombe cells.
Figure 5A and 5B are photographs of yeast colonies formed by S. pombe cells transformed with pART3 plasmid, grown at 25°C and 37°C
respectively.
Figures 6A and 6B are photographs of yeast colonies formed by S. pombe cells transformed with the pARTN-cdc25A plasmid of Example l, grown at 25°C
and 37°C
respectively.
Figures 7A and 7B are photographs of yeast colonies formed by S. pombe cells transformed with the pARTN-cdc25B plasmid of Example 1, grown at 25°C
and 37°C
respectively.
Figures 8A and 8B are photographs of yeast colonies formed by S. pombe cells transformed with the pARTN-cdc25C plasmid of Example 1, grown at 25°C
and 37°C
respectively.

2163~~4 Detailed Description of the Invention In dividing eukaryotic cells, circuits of regulatory proteins oversee both the initiation and completion of the major transitions of both the meiotic and mitotic cell-cycles. These regulatory networks guarantee that the successive events of each cell-cycle occur in a faithful and punctual manner. For example, mitosis cannot begin until the cell has grown sufficiently and replicated its genome accurately. Likewise, cell division cannot ensue until the mitotic spindle has distributed the chromosomes equally to both daughter cells.
The present invention makes available assays and reagents for identifying anti-mitotic and anti-meiotic agents. As described herein, anti-mitotic agents can be identified, in one embodiment of the present assay, through their ability to rescue an otherwise hyper-mitotic cell from mitotic catastrophe by inhibiting the activity of at least one regulatory protein of the cell-cycle which acts as a mitotic activator. The term hyper-mitotic cell denotes a cell having 1 S an impaired cell-cycle checkpoint which can cause premature progression of the cell though at least a portion of the cell-cycle and thereby results in inhibition of proliferation of the cell.
The impaired checkpoint of the hyper-mitotic cell would otherwise act as a negative regulator of downstream mitotic events. Impairment of such a negative regulator consequently allows the cell to proceed aberrantly toward subsequent mitotic stages and ultimately inhibits faithful proliferation of the cell. In the presence of an agent able to inhibit a mitotic activator, progression of the hyper-mitotic cell through the cell-cycle can be slowed to enable the cell to appropriately undergo mitosis and proliferate with fidelity. In general, it will be expected that in order to detect an anti-mitotic agent in the present assay using a hyper-mitotic cell, the agent must inhibit a mitotic activator whose operation in the cell-cycle is sufficiently connected to the impaired checkpoint that the cell is prevented by the anti-mitotic agent from committing to the otherwise catastrophic events of prematurely passing the checkpoint. It is clear that an anti-mitotic agent effective at rescuing the hyper-mitotic cell in the present assay can do so by acting directly on the mitotic activator such as, for example, a phosphatase inhibitor might be expected to do to a cdc25 homolog.
Alternatively, the anti-mitotic agent may exert its effect by preventing the activation of the mitotic activator, as, for example, inhibiting the phosphorylation step which activates cdc25 as a phosphatase, or inhibiting the activity of the cdc2 kinase with regard to other potential protein substrates.
In another embodiment of the present assay, an anti-mitotic agent can be identified by its ability to induce mitosis in an otherwise hypo-mitotic cell by inhibiting the activity of at least one regulatory protein of the cell-cycle which acts as a negative regulator of mitosis.
The term hypo-mitotic cell refers to a cell which has an impaired checkpoint comprising an overly-active negative mitotic regulator which represses progression of the cell through at WO 94128914 ~ PCTIUS94106365 _,_ least a portion of the cell-cycle. In the presence of an agent able to inhibit the activity of the negative regulator, inhibition of the cell-cycle is overcome and the cell can proliferate at an increased rate relative to the untreated hypo-mitotic cell. As with the hyper-mitotic system above, it will generally be expected that an anti-mitotic agent detected in the hypo-mitotic system acts at, or sufficiently close to, the overly-active negative regulator so as to reduce its inhibitory effect on the cell-cycle.
In yet another embodiment of the present invention, anti-meiotic agents can be identified in a manner analogous to the anti-mitotic assay above, wherein faithful meiosis of either a hyper-meiotic or hypo-meiotic cell is measured in the presence and absence of a candidate agent. As above, the terms hyper-meiotic and hypo-meiotic refer to impaired meiotic checkpoints which are respectively of either diminished activity or enhanced activity relative to the normal meiotic cell.
1 S The present assay provides a simple and rapid screening test which relies on scoring for positive proliferation as indicative of anti-mitotic activity. One advantage of the present assay is that while direct inhibition of growth can be caused by any toxic compound added to a proliferating cell culture, growth stimulation in the present assay will only be achieved upon specific inhibition of a mitotic activator where the assay comprises a hyper-mitotic cell, or upon inhibition of a negative mitotic regulator where the assay comprises a hypo-mitotic cell. In an analogous manner, positive meiotic progression can be utilized in the present assay as indicative of anti-meiotic activity of the candidate agent.
Other advantages of the present assays include the ability to screen for anti-mitotic and anti-meiotic activity in vivo, as well as the amenity of the assay to high through-put analysis. Anti-mitotic agents identified in the present assay can have important medical consequences and may be further tested for use in treating proliferative diseases which include a wide range of cancers, neoplasias, and hyperplasias, as well as for general or specific immunosuppression, such as through inhibition of the proliferation of lymphocytes.
In addition, the present assay can be used to identify both anti-mitotic and anti-meiotic agents which can be used in the treatment of pathogenic infections such as fungal infections which give rise to mycosis. Anti-mitotic and anti-meiotic agents identified in the present assay may ' also be used, for example, in birth control methods by disrupting oogenic pathways in order to prevent the development of either the egg or sperm, or by preventing mitotic progression of a fertilized egg.
With regard to the hyper-mitotic cell and hypo-mitotic cell of the present assay, WO 94128914 PCTlUS94106365 21635~9~
_8_ impairment of the negative regulatory checkpoint can be generated so as to be either continual or conditional. A conditional impairment permits the checkpoint to be normatively operational under some conditions such that the cell may proliferate and be maintained by cell culture techniques; and be rendered inoperative, or alternatively hyper-operative, under S other conditions. In the instance of the hyper-mitotic cell, the impaired checkpoint is effectively inoperative to an extent that the impairment allows aberrant mitosis to occur which concludes in mitotic catastrophe (e.g. cell death). Conversely, the hypo-mitotic cell can be generated by an impaired checkpoint which is effectively hyper-operative and results in inhibition of the cell-cycle. A continual impairment, on the other hand, is one that is ever-present and which allows proliferation of the cell under conditions where there is no need to halt the cell at that checkpoint; but, in the instance of the hyper-mitotic cell, results in mitotic catastrophe under conditions where the cell-cycle must be halted, such as in the presence of DNA synthesis inhibitors or DNA damaging agents.
The impaired checkpoint can be generated, for example, by molecular biological, genetic, and/or biochemical means. The checkpoint to be impaired can comprise a regulatory protein or proteins which control progression through the cell-cycle, such as those which control the G2/M transition or the G 1 /S transition. Extensive genetic and biochemical analysis of these pathways (see, for example, Molecular Biology of the Fission Yeast, eds Nasi et al., Academic Press, San Diego, 1989) has led to the ability to manipulate the control of mitosis through loss-of function and gain-of function mutations and by plasmid overexpression, as well as by exposure of the cell to certain chemicals. The checkpoint impairment can be, for example, the result of directly altering the effective activity of a regulatory protein at the checkpoint (i.e. by altering its catalytic activity and/or concentration), or indirectly the result of modifying the action of another protein which is upstream of the checkpoint but which modulates the action of regulatory proteins at the checkpoint. For instance, various mutants have been isolated which are able to escape specific cell-cycle control circuits and progress inappropriately to the next cell-cycle stage and can be used to generate the hyper-mitotic cell. In a similar manner, mutants have been isolated which are unable to pass a specific cell-cycle checkpoint and are prevented from progressing to the next cell-cycle stage, and provide the basis for the hypo-mitotic cell of the present assay.
Genetic studies in eukaryotic systems, including mammalian and fungi, have identified several genes that are important for the proper timing of mitosis.
For instance, in the fission yeast S. pombe, genes encoding regulators of cell division have been extensively characterized (for review see MacNeil et al. ( 1989) Curr. Genet. 16:1 ). As set out above, ~'1 ~3~.
initiation of mitosis in fission yeast correlates with activation of the cdc2 protein kinase.
cdc2 is a component of M phase promoting factor (MPF) purified from frogs and starfish, and homologs of cdc2 have been identified in a wide range of eukaryotes, suggesting that cdc2 . plays a central role in mitotic control in all eukaryotic cells (Norbury et al. (1989) Biochem.
S Biophys. Acta 989:85). For purposes of the present disclosure, the term "cdc2" or "cdc protein kinase" is used synonymously with the recently adopted "cyclin-dependent kinase"
(cdk) nomenclature. Furthenmore as used herein, the term cdc2 is understood to denote members of the cyclin-dependent kinase (cdk) family. Representative examples of cdc protein kinases include cdc2-SP, cdc28 (S. Cerevisiae), cdk2-XL, cdc2-HS and cdk2-HS, where "HS" designates homosapiens, SP designates S. pombe, and "XL" designates Xenopus Laevis. As set out above, the switch that controls the transition between the inactive cdc2/cyclin B complex (phosphorylated on Try-15 and Thr-14) present during S-G2-prophase and the active form of the cdc2/cyclin B kinase (dephosphorylated on Try-15 and Thr-14) present at metaphase is believed to correspond to a change in the relative activities of the opposing kinases and phosphatase(s) that act on the sites. Given that many regulatory pathways appear to converge on cdc protein kinases, as well as their activating role at both G 1 /S and G2/M transitions, the hyper-mitotic cell of the present assay can be employed to develop inhibitors specific for particular cdc protein kinases.
Regulatory pathways which feed into and modulate the activity of a cdc protein kinase can be manipulated to generate either the hyper-mitotic or hypo-mitotic cell of the present assay. For example, the inhibitory phosphorylation of cdc2 is mediated by at least two tyrosine kinases, initially identified in fission yeast and known as weel and mild (Russell et al. ( 1987) Cell 49:559; Lundgren et al. ( I 991 ) Cell 64:111;
Featherstone et al.
'_'S (1991) Nature 349:808; and Parker et al. (1991) EMBO 10:1255). These kinases act as mitotic inhibitors, overexpression of which causes cells to arrest in the G2 phase of the cell-cycle. For instance, overexpression of wee 1 has been shown to cause intense phosphorylation of cdc2 (cdc28 in budding yeast) which results in cell-cycle arrest.
Conversely, loss of function of weel causes advancement of mitosis and cells enter mitosis at approximately half the normal size, whereas loss of weel and mikl function causes grossly premature initiation of mitosis, uncoupled from all checkpoints that normally restrain cell division. Thus, weel and mikl each represent suitable regulatory proteins which could be impaired to generate either the hyper-mitotic or hypo-mitotic cell of the present assay.
Furthermore, it is apparent that enzymes which modulate the activity of the weel or mikl kinases can also be pivotal in controlling the precise timing of mitosis.
For example, the level of the nim 1 /cdrl protein, a negative regulator of the wee 1 protein kinase, can have a 2163~~4 _1o_ pronounced impact on the rate of mitotic initiation, and nim 1 mutants have been shown to be defective in responding to nutritional deprivation (Russel et al. ( 1987) Cell 49:569; and Feilotter et al. (1991) Genetics 127:309). Over-expression of niml (such as the S. pombe op-niml mutant) can result in inhibition of the wee! kinase and allow premature progression into mitosis. Loss of niml function, on the other hand, delays mitosis until the cells have grown to a larger size. In like manner, mutation in the stfl gene has also been shown to relieve regulation of mitotic progression in response to DNA synthesis inhibition.
Loss-of function strains, such as wee!-50, mikl: : ura, or stfl -1 (Rowley et al. ( 1992) Nature 356:353), are well known. In addition, each of the wee!, mild, and niml genes have been cloned (see for example Coleman et al. ( 1993) Cell 72:919; and Feilotter et al. ( 1991 ) Genetics 127:309), such that disruption of wee! and/or mikl expression or over-expression of nim 1 can be carried out to create the hyper-mitotic cell of the present assay. In a similar fashion, over-expression of wee! and/or mikl or disruption of niml expression can be utilized to generate the hypo-mitotic cell of the present assay. Furthermore, each of these negative mitotic regulators can also be a potential target for an anti-mitotic agent scored for using the hypo-mitotic cell of the present assay.
Acting antagonistically to the wee 1 /mik 1 kinases, genetic and biochemical studies have indicated that the cdc25 protein is a central player in the process of cdc2-specific dephosphorylation and crucial to the activation of the cdc2 kinase activity.
In the absence of cdc25, cdc2 accumulates in a tyrosine phosphorylated state and can cause inhibition of mitosis. The phosphatase activity of cdc25 performs as a mitotic activator and is therefore a suitable target for inhibition by an anti-mitotic agent in the present assay.
It is strongly believed that this aspect of the mitotic control network is generally conserved among eukaryotes, though the particular mode of regulation of cdc25 activity may vary somewhat from species to species. Homologs of the fission yeast cdc25 have been identified in the budding yeast S. cerevisiae (Millar et al. (1991) CSH Symp. Quant. Biol.
56:577), humans (Galaktinov et al. (1990) Cell 67:1181; and Sadhu et al. (1989) PNAS 87:5139), mouse (Kakizuka et al. (1992) Genes Dev. 6:578), Drosophila (Edgar et al. (1989) Cell 57:177; and Glover (1991) Trends Genet. 7:125), and Xenopus (Kumagai et al., (1992) Cell 70:139; and Jessus et al. (1992) Cell 68:323). Human cdc25 is encoded by a multi-gene family now consisting of at least three members, namely cdc25A, cdc25B and cdc25C. As described below, all three homologs are able to rescue temperature-sensitive mutations of the S. Pombe cdc25. Early evidence suggests that these different homologs may have different functions.
For instance, microinjection of anti-cdc25-C antibodies into mammalian cells prevents them from dividing. They appear to arrest in interphase with a flattened morphology, consistent WO 94128914 S' PCTILJS94/06365 z~
with a role for cdc25C in the entry into mitosis. On the contrary, microinjection of antibodies to cdc25A results in a rounded-up mitotic-like state, suggesting that the different homologs may have distinct functions and represent an additional level of complexity to the control of . M-phase onset by cdc25 in higher eukaryotes. Comparison of the human cdc25's with each other and with cdc25 homologs from other species has been carried out.
Comparison of cdc25A with cdc25C demonstrates a 48% identity in the 273 C-terminal region between the two proteins; and comparison between cdc25B and cdc25C reveals a 43% identify.
The Drosophila cdc25 homolog "string" shares 34.5% identity to cdc25A in a 362 amino acid region and 43.9% in an 269 amino acid region with cdc25B. S. Pombe cdc25 is also related to the human cdc25's, but to a lesser extent. Interestingly, the overall similarity between different human cdc25 proteins does not greatly exceed that between humans and such evolutionary distinct species as Drosophila. Biochemical experiments have demonstrated that bacterially produced cdc25 protein from Drosophila and human activates the histone H 1 kinase activity of cdc2 in Xenopus or starfish extracts (Kumagai et al. ( 1991 ) Cell 64:903;
and Strausfield et al. ( 1991 ) Nature 3 51:242).
If the cdc25 phosphatase activity is the desired target for development of an anti-mitotic agent, it may be advantageous to chose the hyper-mitotic cell of the present assay so as to more particularly select for anti-mitotic agents which act directly or indirectly on cdc25.
As set out above, it will generally be expected that in order to score for an anti-mitotic agent in an assay relying on a hyper-mitotic cell, the inhibited mitotic activator (e.g. edc25) must be sufficiently connected to the abherent checkpoint so as to rescue the cell before it concludes in mitotic catastrophe. Furthermore, the hyper-mitotic cell of the present assay can be generated by manipulation of the cell in which a cdc25 homolog is endogenously expressed, as for example, by generating a wee 1 mutation (a "wee" phenotype), or by exposure of the cell to 2-aminopurine or caffeine after a y-radiation induced G2 arrest.
Alternatively, the cdc25 gene from one species or cell type can be cloned and subsequently expressed in a cell to which it is not endogenous but in which it is known to rescue lack-of function mutations of the endogenous cdc25 activity. For example, the exogenous cdc25, such as a human cdc25, could be expressed in an hyper-mitotic Schizosaccharomyces cell, such as an S
pombe cell like the temperature-sensitive wee I -50 mutant. It may be possible to take advantage of the structural and functional differences between the human cdc25 phosphatases to provide anti-mitotic agents which selectively inhibit particular human cell types. In a similar manner, it may be feasible to develop cdc25 phosphatase inhibitors with the present assay which act specifically on pathogens, such as fungus involved in mycotic infections, without substantially inhibiting the human homologs.

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The cdc2 activating kinase (CAK) represents yet another potential target for inhibition by an anti-mitotic agent which could be scored for using the hyper-mitotic cell of the present assay. Recent evidence indicates that many, if not all, of the cdc protein kinases require cyclin binding as well as phosphorylation at Thr-161 (Thr-161 of cdc2-HS; Thr-167 of cdc-2SP; Thr-169 of cdc28; and Thr-160 of cdk2-HS) for activation in vivo. CAK is believed to direct phosphorylation of Thr-161 in a cyclin-dependent manner and to act as a mitotic activator. Inhibition of CAK by a candidate agent may offset the effect of a hyper-mitotic checkpoint impairment which would otherwise have led to premature activation of a cdc protein kinase (e.g. as a wee 1 deficient mutant would). In addition, CAK
itself represents a possible site of impairment to generate the hyper-mitotic cell of the present assay.
Overexpression of CAK can lead to premature activation of a cdc protein kinase and cause the cell to conclude in mitotic catastrophe.
Other checkpoints which could be impaired to generate the hyper-mitotic and hypo-mitotic systems have been identified by examination of mitotic events in cells treated in a manner which disrupts DNA synthesis or DNA repair. Radiation-induced arrest is one example of a checkpoint mechanism which has been used to identify both negative and positive regulators of mitosis. In this instance, mitosis is delayed until the integrity of the genome is checked and, as far as possible, restored. Checkpoint controls also function to delay mitosis until DNA synthesis is complete. The observation of cell-cycle arrest points indicate that the regulation of progression into mitosis in response to both DNA damage and the DNA synthesis requires components of the mitotic control. For example, analysis of radiation-sensitive mutations in budding yeast have identified a number of defective regulatory proteins which can prevent the arrest of the cell-cycle in response to DNA damage and are therefore potential candidates for impairment to generate the hyper-mitotic or hypo-mitotic cell of the present assay. By way of illustration, a number of genes involved in this mitotic feedback control have been identified, and include the rad9, rad 17, rad24, mec 1, mec2 and mec3 genes (Weinert et al. (1988) Science 241:317). All six genes have been shown to be negative regulators of cell-cycle progression and act in response to damaged DNA. Two genes, mecl and mec2, are also involved in arresting the cell-cycle in response to unreplicated DNA.
The response to DNA damage has also been investigated in the fission yeast S
pombe. Mutations in a number of genes have been identified which allow cells with damaged or unreplicated DNA to enter mitosis. For example, the HUS 12 and HUS 16 genes have been implicated as negative regulators of mitosis which respond to unreplicated DNA, while RAD21 is a negative regulator sensitive to damaged DNA. The HUS 14, HUS 17, HUS22, WO 94128914 ~~~ PCT/US94106365 z4 HUS26, RAD 1, RAD3, RAD9 and RAD 17 genes of S. Pombe each appear to be negative regulators of mitosis which are able to respond to either unreplicated or damaged DNA.
(Rowley et al. ( 1992) EMBO 11:1343; and Enoch et al ( 1991 ) CSH Symp. Quant.
Biol.
56:409) Recently, mutations in the S. cerevisiae genes BUB and MAD have been isolated which fail to arrest in mitosis with microtubule-destabilizing drugs. (Hayt et al. ( 1991 ) Cell 66:507; and Li et al. (1991) Cell 66:519). The S. cerevisiae cell can also be affected by a number of environmental cues. One such effector is the a-mating factor which induces G 1 arrest. Mutants in the FUS3 or FAR1 genes fail to arrest in G1 in response to a-factor.
While mutations in either gene are phenotypically similar, they affect different regulatory pathways. For example, the FUS3 gene has been cloned and exhibits strong sequence similarity to the serine/threonine family of protein kinases (Goebl et al. ( 1991 ) Curr. Opin.
Cell Biol. 3:242).
In the fungus Aspergillus nidulans, the bimE gene is believed to code for a negative regulator of mitosis that normally functions to prevent mitosis by controlling expression of a putative mitotic inducer, nimA. The absence of bimE function is believed to override cell-cycle control systems normally operative to prever.e chromosome condensation and spindle formation from occurring during interphase. Temperature sensitive mutants of the bimE
gene, such as the bimE7 mutant, allow cells with unreplicated DNA to prematurely enter mitosis (Osmani et al. (1988) Cell 52:241) and can be lethal phenotypes useful as hyper-mitotic cells of the present assay.
Checkpoints, and mutations thereof, have been identified in mammalian cells as well, and can be used to generate the hyper-mitotic and hypo-mitotic cells of the present assay.
For instance, uncoupling of mitosis from completion of DNA replication has been reported in mammalian cells in response to drug treatment and mutation. In mammalian cells, as in other eukaryotic cells, DNA damage caused by mild X-ray irradiation can block passage through two cell-cycle checkpoints, the restriction point (G1/5) and entry into mitosis (G2/M) (Little et al. (1968) Nature 218:1064; Nagasawa et al. (1984) Radiation Res 97:537;
and Murray ( 1992) Nature 359:599). The AT gene(s), p53 and GADD45 are among genes which have been identified as critical to negative regulation of mitosis by cell-cycle checkpoints (Kaastan et al. (1992) ~ 71:587; Hartwell (1992) ~ 71:543; and Murray (1992) Nature 359:599) and can be utilized in the present assay to generate a hyper-mitotic cell or a hypo-mitotic cell depending on whether the impairment is brought about by disruption of expression, inhibition of activity, or by overexpression. Additionally, a temperature-sensitive mutation in the WO 94128914 . PCT/US94106365 213524 -~4-mammalian RCC 1 (repressor of chromosome condensation) gene can cause cultured hamster cells to cease DNA replication and enter mitosis prematurely when they are shifted up to the nonpermissive temperature during S. phase. Relatives of RCC 1 have also been identified in yeast (i.e. pim 1 ) and Drosophila, and both genes can complement the mammalian RCC 1 mutation, further suggesting that certain checkpoint mechanisms, like cdc2 regulation of the cell-cycle, are conserved across diverse phyla.
Many of the regulatory proteins involved in the progression of a cell through meiosis have also been identified. Because of the commonalty of certain mitotic and meiotic pathways, several mitotic regulatory proteins or their homologs, such as cdc protein kinases, cyclins, and cdc25 homologs, also serve to regulate meiosis. For example, cell division cycle mutants defective in certain mitotic cell-cycle events have been tested for sporulation at semi-restrictive temperatures (Gralbert et al. ( 1991 ) Curr Genet 20:199). The mitotic defective mutants cdcl0-129, cdc20-M10, cdc21-M6B, cdc23-M36 and cdc24-M38 formed four-spored asci but with low efficiency. Mutants defective in the mitotic initiation genes cdc2, cdc25 and cdc 13 were blocked at meiosis II, though none of the wee 1-50, ddh.
nim 1 + and winl+ alleles had any affect on sporulation, suggesting that their interactions with cdc25 and cdc2 are specific to mitosis in yeast. Other regulatory genes and gene products which can be manipulated to form the hyper- or hypo-meiotic cells of the present invention include rec 102, spol3, cutl, cut2, IME1, MAT, RME1, cdc35, BCY1, TPK1, TPK2, TPK3, spdl, spd3, spd4, spo50, spo5l, and spo53. As above, the hyper- or hypo-meiotic cells can be generated genetically or chemically using cells to which the intended target of the anti-meiotic agent is endogenous, or alternatively, using cells in which the intended target is exogenously expressed.
In addition, certain meiotic regulatory proteins are able to rescue loss-of function mutations in the mitotic cell-cycle. For example, the Drosophila meiotic cdc25 homolog, "twine", is able to rescue mitosis in temperature-sensitive cdc25 mutants of fission yeast.
Thus, anti-meiotic agents can be identified using hyper- or hypo-meiotic cells, and in some instances, hyper- or hypo-mitotic cells.
It is also deemed to be within the scope of this invention that the hyper- and hypo-proliferative cells of the present assay, whether for identifying anti-mitotic or anti-meiotic agents, can be generated so as to comprise heterologous cell-cycle proteins (i.e. cross-species expression). As exemplified above in the instance of cdc25, cell-cycle proteins from one species can be expressed in the cells of another and have been shown to be able to rescue 3~~~t loss-of function mutations in the host cell. In addition to those cell-cycle proteins which are ideally to be the target of inhibition by the candidate agent, cell-cycle proteins which interact with the intended inhibitor target can also be expressed across species. For example, in an hyper-proliferative yeast cell in which a human cdc25 (e.g. exogenously expressed) is the intended target for development of an anti-mitotic agent, a human cdc protein kinase and human cyclin can also be expressed in the yeast cell. Likewise, when a hypo-proliferative yeast expressing human wee 1 is used, a human cdc protein kinase and human cyclin with which the human cdc25 would interact can be used to replace the corresponding yeast cell-cycle proteins. To illustrate, a triple cln deletion mutant of S. Cerevisae which is also conditionally deficient in cdc28 (the budding yeast equivalent of cdc2) can be rescued by the co-expression of a human cyclin and human cdc2 proteins, demonstrating that yeast cell cycle machinery can be at least in part replaced with corresponding human regulatory proteins. Roberts et al. (1993) PCT Publication Number WO 93/06123. In this manner, the reagent cells of the present assay can be generated to more closely approximate the natural interactions which a particular cell-cycle protein might experience.
Manipulation of these regulatory pathways with certain drugs, termed here "hyper-mitotic agents", can induce mitotic aberrations and result in generation of the hyper-mitotic cell of the present assay. For instance, caffeine, the protein kinase inhibitors 2-aminopurine and 6-dimethylaminopurine, and the protein phosphatase inhibitor okadaic acid can cause cells that are arrested in S phase by DNA synthesis inhibitors to inappropriately enter mitosis (Schlegel et al. (1986) Science 232:1264; Schlegel et al. (1987) PNAS 84:9025;
and Schlegel et al. ( 1990) Cell Growth Differ. 1:171 ). Further, 2-aminopurine is believed to be able to override a number of cell-cycle checkpoints from G1, S phase, G2, or mitosis.
(Andreassen et al. (1992) PNAS 89:2272; Andreassen et al. (1991) J. Cell Sci. 100:299, and Steinmann et al.
(1991) PNAS 88:6843). For example, 2-aminopurine permits cells to overcome a block induced by y-irradiation. Additionally, cells continuously exposed to 2-aminopurine alone are able to exit S phase without completion of replication, and exit mitosis without metaphase, anaphase, or telophase events.
In an analogous manner, hypo-mitotic agents, such as a phosphatase inhibitor, can be utilized to chemically induce impairment of one or more regulatory pathways to produce the hypo-mitotic cell of the present assay. Likewise, hyper-meiotic or hypo-meiotic agents can be employed to chemically generate the appropriate reagent cell for identifying anti-meiotic agents in the present assay.
To aid in the facilitation of mitotic catastrophe in the hyper-mitotic cell it may be desirable to expose the cell to an agent (i.e. a chemical or environmental stimulus) which ordinarily induces cell-cycle arrest at that checkpoint. Inappropriate exit from the chemically- or environmentally-induced arrested state due to the impairment of the negative regulatory checkpoint can ultimately be lethal to the cell. Such arresting agents can include exposure to DNA damaging radiation or DNA damaging agents; inhibition of DNA
synthesis and repair using DNA polymerase inhibitors such as hydroxyurea or aphidicolin;
topoisomerase inhibitors such as 4'-dimethly-epipodophyllotoxin (VM-26); or agents which interfere with microtubule-assembly, such as Nocadazole and taxol. By way of example, BHK and HeLa cells which receive 250 rads of y radiation have been shown to undergo G2 I 0 arrest that was reversed without further treatment within 4-5 hours.
However, in the presence of either caffeine, 2-aminopurine, or 6-dimethyl-aminopurine, this mitotic delay was suppressed in both the hamster and human cells, and allowed the cells undergo mitosis before DNA repair had been completed (Steinmann et al. (1991) PNAS 88:6843).
Additionally, in certain cells, nutritional status of the cell, as well as mating factors, can cause arrest of the I 5 normal cell during mitosis.
The present assay can be used to develop inhibitors of fungal infections. The most common fungal infections are superficial and are presently treated with one of several topical drugs or with the oral drugs ketoconazole or griseofulvin. The systemic mycoses constitute 20 quite a different therapeutic problem. These infections are often very difficult to treat and long-term, parenteral therapy with potentially toxic drugs may be required.
The systemic mycoses are sometimes considered in two groups according to the infecting organism. The "opportunistic infections" refer to those mycoses -candidiasis, aspergillosis, cryptococcosis, and phycomycosis- that commonly occur in debilitated and immunosuppressed patients.
25 These infections are a particular problem in patients with leukemias and lymphomas, in people who are receiving immunosuppressive therapy, and in patients with such predisposing factors as diabetes mellitus or AIDS. Other systemic mycoses -for example, blastomycosis, histoplasmosis, coccidiodomycosis, and sporotrichosis- tend to have a relatively low incidence that may vary considerably according to geographical area.
To develop an assay for anti-mitotic or anti-meiotic agents having potential therapeutic value in the treatment of a certain mycotic infection, a yeast implicated in the infection can be used to generate the appropriate reagent cell of the present assay. For example, the hyper-mitotic or hypo-mitotic cell can be generated biochemically as described above, or engineered, as for example, by screening for radiation-sensitive mutants having impaired checkpoints. Additionally, a putative mitotic regulator of the mycotic yeast, such as a cdc25 homolog, can be cloned and expressed in a heterologous cell which may be easier to _ ~'163~'2~
manipulate or facilitate easier measurement of proliferation, such as member of the Schizosaccharomyces genus like S. pombe.
By way of illustration, the present assays can be used to screen for anti-mitotic and anti-meiotic agents able to inhibit at least one fungus implicated in such mycosis as candidiasis, aspergillosis, mucormycosis, blastomycosis, geotrichosis, cryptococcosis, chromoblastomycosis, coccidioidomycosis, conidiosporosis, histoplasmosis, maduromycosis, rhinosporidosis, nocaidiosis, para-actinomycosis, penicilliosis, monoliasis, or sporotrichosis.
For example, if the mycotic infection to which treatment is desired is candidiasis, the present assay can comprise either a hyper-mitotic or hypo-mitotic cells generated directly from, or with genes cloned from, yeast selected from the group consisting of Candida albicans, Candida stellatoidea, Candida tropicalis, Candida parapsilosis, Candida krusei, Candida pseudotropicalis, Candida guillermondii, and Candida rugosa. Likewise, the present assay can be used to identify anti-mitotic and anti-meiotic agents which may have therapeutic value in the treatment of aspergillosis by making use of yeast such as Aspergillus fumigatus, Aspergillus,flavus, Aspergillus niger, Aspergillus nidulans, or Aspergillus terreus. Where the mycotic infection is mucormycosis, the yeast can be selected from a group consisting of Rhizopus arrhizus, Rhizopus oryzae, Absidia corymbifera, Absidia ramosa, and Mucor pusillus. Other pathogens which can be utilized in the present assay include Pneumocystis carinii and Toxoplasma gondii.
Agents to be tested for their ability to act as anti-mitotic and/or anti-meiotic agents in the present assay can be those produced by bacteria, yeast or other organisms, or those produced chemically. The assay can be carried out in any vessel suitable for the growth of the cell, such as microtitre plates or petri dishes. As potent inhibitors mitosis and/or meiosis can fully inhibit proliferation of a cell, it may be useful to perform the assay at various concentrations of the candidate agent. For example, serial dilutions of the candidate agents can be added to the hyper-mitotic cell such that at at least one concentration tested the anti-mitotic agent inhibits the mitotic activator to an extent necessary to adequately slow the progression of the cell through the cell-cycle but not to the extent necessary to inhibit entry into mitosis all together. In a like manner, where the assay comprises a hypo-mitotic cell, serial dilutions of a candidate agent can be added to the cells such that, at at least one concentration, an anti-mitotic agent inhibits a negative mitotic regulator to an extent necessary to adequately enhance progression of the cell through the cell-cycle, but not to an extent which would cause mitotic catastrophe.
Quantification of proliferation of the hyper-mitotic cell in the presence and absence of a candidate agent can be measured with a number of techniques well known in the art, including simple measurement of population growth curves. For instance, where the assay involves proliferation in a liquid medium, turbidimetric techniques (i.e.
absorbence/transmittance of light of a given wavelength through the sample) can be utilized.
For example, in the instance where the reagent cell is a yeast cell, measurement of absorbence of light at a wavelength between 540 and 600nm can provide a conveniently fast measure of cell growth.
Likewise, ability to form colonies in solid medium (e.g. agar) can be used to readily score for proliferation. Both of these techniques, especially with respect to yeast cells, are suitable for high through-put analysis necessary for rapid screening of large numbers of candidate agents. In addition, the use of solid media such as agar can further aid in establishing a serial dilution of the candidate agent. For example, the candidate agent can be spotted on a lawn of reagent cells plated on a solid media. The diffusion of the candidate agent through the solid medium surrounding the site at which it was spotted will create a diffusional effect. For anti-mitotic or anti-meiotic agents scored for in the present assay, a halo of cell growth would be expected in an area which corresponds to concentrations of the agent which offset the effect of the impaired checkpoint, but which are not so great as to over-compensate for the impairment or too little so as to be unable to rescue the cell.
To further illustrate, other proliferative scoring techniques useful in the present assay include measuring the mitotic index for untreated and treated cells; uptake of detectable nucleotides, amino acids or dyes; as well as visual inspection of morphological details of the cell, such as chromatin structure or other features which would be distinguishable between cells advancing appropriately through mitosis and cells concluding in mitotic catastrophe or stuck at certain cell-cycle checkpoint. In the instance of scoring for meiosis, morphology of the spores or gametes can be assessed. Alternatively, the ability to form a viable spore of gamete can be scored as, for example, measuring the ability of a spore to re-enter negative growth when contacted with an appropriate fermentable media.
To test compounds that might specifically inhibit the human cdc25A, cdc25B or cdc25C gene products, the genes were introduced into the genome of an S. pombe strain which was engineered to be conditionally hyper-mitotic. Three linear DNA
fragments were constructed, each carrying one of the three human cdc25A, B or C genes under the control of an S. pombe promoter, and flanked by nucleic acid sequences which allow integration of the DNA into the S. pombe genome. The cdc25-containing DNA fragments are then used to transform an appropriate S. pombe strain. For example, in one embodiment, the expression of the human cdc25 gene is driven by the strong adh promoter and the flanking sequences of the fragment contain the ura4 gene to allow integration of the fragment at the ura4 locus by WO 94/28914 ~~~ PCT/LTS94/06365 - 3~',2~
homologous recombination (Grimm et al. (1988) Molec. gen. Genet 81-86). The S:
pombe strain is a wee 1 temperature-sensitive mutant which becomes hyper-mitotic at temperatures above 36 °C, and carries a wild-type ura4 gene in which the cdc25 DNA
fragment can be integrated.
The human cdc25A gene has been previously cloned (see Galaktinov et al. ( 1991 ) Cell 67:1 I 81 ). The sequence of the cdc25A gene containing the open reading frame is shown in Seq. ID No. 1, and is predicted to encode a protein of 523 amino acids (Seq. ID No. 2). A

2.0 kb Ncol-KpnI fragment encoding amino acids 1-523 of human cdc25A was subcloned into a NcoI-KpnI-(partially) digested pARTN expression vector, resulting in the pARTN-cdc25A construct harboring human cdc25A cDNA in sense orientation to the constitutive adh promoter. The S. Pombe autonomously replicating pARTN vector is derived from pART3 1 S (McLeod et al. ( 1987) EMBO 6:729) by ligation of a NcoI linker (New England Biolabs) into the SmaI site.
A 2.3 kb DNA fragment corresponding to the adh promoter and amino acids 1-523 of the human cdc25A gene, was isolated by digesting the pARTN-cdc25A plasmid with HindIII
and Asp718. While HindIII is sufficient to isolate the adh promoter/human cdc25A gene fragment from the plasmid, we also used Asp718 to cut the close migrating 2.2 kb HindIII-HindII1 S. cerevisiae LEU2 gene in two smaller fragments which makes isolation of the cdc25A fragment easier.
?5 The HindIII/HindIII fragment was then blunt ended with Klenow enzyme and dNTPs (see Molecular Cloning. A Laboratory Manual Zed, eds. Sambrook et al., CSH
Laboratory Press: 1989) and ligated into a pKS-/ura4 plasmid previously digested with StuI and dephosphorylated with alkaline phosphatase. Massive amounts of the recombinant plasmid were prepared, and a 4.1 kb DNA fragment corresponding to "5'-half ura4-adh promoter cdc25A-3'-half ura4" (see Figure 1 ) was isolated.
The human cdc25B gene has been previously cloned (see Galaktinov et al. ( 1991 ) Cell 67: i I 81 ). The sequence of the cdc25B gene containing the open reading frame is shown in Seq. ID. No. 3, and is predicted to encode a protein of 566 amino acids (Seq. ID No. 4). A
2.4 kb SmaI fragment from the p4x 1.2 plasmid (Galaktinov et al., supra) encoding amino acids 32-566 was subcloned into a SmaI-digested pART3 vector, resulting in the pARTN-cdc25B vector containing the human cdc25B cDNA. While the site of initiation of translation is not clear (there is no exogenous ATG 5' to the SmaI cloning site in the cdc25B
open reading frame) we speculate that the first ATG corresponds to the Met-59 of the human cdc25B open reading frame, or alternatively, an ATG at an NdeI site of pART3.
In any event, the pARTN-cdc25B plasmid has been shown to be capable of transforming S. pombe cells and able to rescue temperature-sensitive mutations of the yeast cdc25 gene (Galaktinov et al., supra).
As above, a 2.7 kb DNA fragment, corresponding to the adh promoter and amino acids 32-566 of the human ede25B gene, was isolated by digesting pARTN-cdc25B
with HindIII and Asp718. The HindIII/HindIII cdc25B fragment was blunt ended with Klenow enzyme and dNTPs, and ligated into a pKS-/ura4 vector previously digested with StuI and dephosphorylated with alkaline phosphatase. A 4.4 kb DNA fragment corresponding to "5' half ura4-adh promoter-cdc-25B-3'-half ura4" (see Figure 2) was isolated.
The human cdc25C gene has been previously cloned (see Sadhu et al. (1990) PNAS
87:115139; and Hoffmann et al. (1993) EMBO 12:53). The sequence of the cdc25C
gene containing the open reading frame is shown in Seq. ID No. 5, and is predicted to encode a protein of 473 amino acids (Seq. ID No. 6). Beginning with the pGEX-2T6-cdc25 plasmid (Hoffmann et al., supra) a 1.8 kbp DNA fragment corresponding to amino acids 1-473 of the human cdc25C gene was isolated digestion with BamHI and by partial digestion with NdeI
(i.e., there is a NdeI site in the cdc25C gene). This fragment was ligated into a pART3 vector previously digested with NdeI and BamHI, resulting in the plasmid pART3-cdc25C
which contained the amino acids 1-473 of the human cdc25C gene under the control of the strong adh promoter (see Figure 3).
A 2.5 kbp fragment corresponding to the adh promoter and amino acids 1-473 of the human cdc25C gene was isolated by digesting pART3-cdc25C with HindIII and Asp718.
The HindIII/HindIII cdc25C fragment was blunt ended with Klenow enzyme and dNTPs, and ligated into a pKS-/ura4 plasmid previously digested with StuI and dephosphorylated with alkaline phosphatase. A 4.3 kbp DNA fragment corresponding to "5'-half ura4-adh promoter cdc25C-3'-half ura4" (see Figure 4) was isolated.

WO 94!28914 ~ ~ ~ 4 PCTIUS94/06365 Each of the cdc25 plasmid constructs pARTN-cdc25A, pARTN-cdc25B, and pART3-cdc25C, as well as the original pART3 plasmid, were used to transform the S. Pombe strain Sp553 (h+N, cdc25-22, wee!-50, leul-32) using well known procedures.
Briefly, cells were grown in YE medium at 25°C until they were in exponential phase (~10~ cells/ml). The cells were then spun down from the media at 3000rpm for 5 minutes, and resuspended in LiCI/TE at a concentration of ~10g cells/ml (LiCI/TE=lOmM Tris, 1mM EDTA, 50 mM
LiCI, Ph 8). The resuspended cells were incubated at room temperature for 10 minutes, then spun again at 3000rpm for 5 minutes, resuspended in LiCI/TE to a concentration of ~5 x 108 cells/ml, and shaken for 30 minutes at 25°C.
To an aliquot of 150p,1 of cells, 500 ng of plasmid DNA and 350~L of PEG/TE
( I OmM Tris, 1 mM EDTA, 50% PEG 4000, Ph 8) was added. The cell/plasmid mixture was then incubated for 30 minutes at 25°C, heat shocked at 42°C for 20 minutes, then spun at 15,000 rpm for 10 seconds after the addition of 0.5 mL of EMM. The cells were resuspended in 0.6 mL EMM, and 0.2 mL aliquots were plated.
Figures 5A and SB illustrate the ability of the pART3 transformed yeast to grow at 25°C and 37°C respectively. As set out above, at the non-permissive temperature of 37°C, both the endogenous wee! and cdc25 activities are impaired such that they mutually off set each other's effects, and the cells are still able to proliferate (pART3 lacks any cdc25 gene).
Figures 6A and 6B (cdc25A), 7A and 7B (cdc25B), and 8A and 8B (cdc25C) demonstrate the effect of expressing a human cdc25 in a yeast "wee"
background. Each of Figures 6A, 7A and 8A show that at the permissive temperature of 25°C
(wee! is expressed) the cells are able to proliferate. However, as illustrated by Figures 6B, 7B
and 8B, shifting the temperature to the non-permissive temperature of 37°C results in mitotic catastrophe.
Microscopic analysis of the yeast cells present on the 37°C plates revealed that the expression of a human cdc25 in a yeast wee background resulted in mitotic catastrophe for the cells.
To provide a more stable transformant and uniform expression of the human cdc25 gene, each of the resulting ura4-cdc25 fragments of Examples 1-3 was used to transform a ura4+ S. pombe strain. As in Example 4, each of the S. pombe strain carried a thermosensitive allele of its own cdc25 gene, such as the cdc25-22 phenotype, so that at non-WO 94/'.8914 216 3 5 2 4 ~TN594106365 _Z~_ permissive temperatures the exogenous cdc2~ is principally responsible for activation of cdc2. In one embodiment, the S. Pombe wee I -~O cdc25-22 ura.f+ strain was traiuformed with a ma4-cdc=3 fragment of Examples 1-3. This particular strain is generally viable at 25°C _as well as the restrictive temperature of 37°C as the loss of endoecnous cdc2~ activity is S recovered by the concomitant loss of wee 1-function at 37°C. However, integration and over expression .of the human cdc2~, as demonstrated in Example 4. can result in a mitotic catastrophic phenotype at 37°C as the weel checkpoint is impaired.
To assay the anti-mitotic activity of various candidate agents, the cells of Example 4 or S are either plated on a solid medium such as EMM plates or suspended in an appropriate vegetative broth such as YE_ 1~ In the instance of plating on a solid ruedium, candidate agents are subsequently blotted onto the plate, and the plate incubated at the non-permissive temperature of 37°C. A
halo of cell growth wilt form surrounding those agents able to at least partially inhibit a mitotic activator which can rescue fl1e otherwise catastrophic cell.
~Jhere growth of the cells is carried out in a vegetative broth, aliquots of cellimedia arc placed in the wells of microtitre plates and serial dilutions of candidate agents are added to the wells. The plates are incubated at 37°C, and the Ag4p for each well measured over time and compared to similar wells of cells/media which lack the candidate agent (e.g.
negative controls}. An increase in absorbence aver time relative to the negative controls 2.5 indicates positive proliferation of the cells and suggests an ability of a particular candidate agent to inhibit a mitotic activator.
.~utyalents Those skilled in the art will recognize, or be able tro 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.
,,Ai ,..-SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT:

(A) NAME: Mitotix, Inc.

(B) STREET: One Kendall Square, Building 600 (C) CITY: Cambridge 1O (D) STATE: MA

(E) COUNTRY: USA

(F) POSTAL CODE (ZIP): 02139 (G) TELEPHONE: (617) 225-0001 (H) TELEFAX: (617) 225-0005 (ii) TITLE OF INVENTION: Assay and Reagents for Identifying Anti-proliferative Agents (iii) NUMBER OF SEQUENCES: 6 (iv) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS

2S (D) SOFTWARE: ASCII (text) (vi) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: US 08/073,383 (B) FILING DATE: 04-JUN-1993 (2) INFORMATION FOR SEQ ID NO: l:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2420 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear 4O (ii) MOLECULE TYPE: cDNA
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 460..2031 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:

2~~3~24 _ CCGTGCCTGT GTCTCGTGGC CACCCGCGCG GCCGCGCCGC

TGGCGCCTGG
AGGGTCCGCA

TTT GCCCGCG GCAGCCGCGT CCTGAACC G GAGTCGTG TTTGTGTTTG

C CG

S

GCC GGTGGCG CGCGGCCGAG CCGGTGTC G GCGGTCGCGC GGGAGGCAGA

G GCGGGGCGGG

TGCGAGGCCG ATG
GG

Met GluLeu GlyPro 1~

Ser Pro AlaPro ArgArgLeu LeuPheAla CysSerProPro ProAla Ser Gln ProVal ValLysAla LeuPheGly AlaSerAlaAla GlyGly Leu Ser ProVal ThrAsnLeu ThrValThr MetAspGlnLeu GlnGly Leu Gly SerAsp TyrGluGln ProLeuGlu ValLysAsnAsn SerAsn 30 Leu Gln IleMet GlySerSer ArgSerThr AspSerGlyPhe CysLeu Asp Ser ProGly ProLeuAsp SerLysGlu AsnLeuGluAsn ProMet Arg Arg IleHis SerLeuPro GlnLysLeu LeuGlyCysSer ProAla Leu Lys ArgSer HisSerAsp SerLeuAsp HisAspIlePhe GlnLeu Ile Asp ProAsp GluAsnLys GluAsnGlu AlaPheGluPhe LysLys S0 Pro Val ArgPro ValSerArg GlyCysLeu HisSerHisGly LeuGln Glu Gly LysAsp LeuPheThr GlnArgGln AsnSerAlaGln LeuGly ~..

Met Leu Ser Ser Asn Glu Arg Asp Ser Ser Glu Pro Gly Asn Phe Ile Pro Leu Phe Thr Pro Gln Ser Pro Val Thr Ala Thr Leu Ser Asp Glu 1O Asp Asp Gly Phe Val Asp Leu Leu Asp Gly Asp Asn Leu Lys Asn Glu Glu Glu Thr Pro Ser Cys Met Ala Ser Leu Trp Thr Ala Pro Leu Val Met Arg Thr Thr Asn Leu Asp Asn Arg Cys Lys Leu Phe Asp Ser Pro Ser Leu Cys Ser Ser Ser Thr Arg Ser Val Leu Lys Arg Pro Glu Arg Ser Gln Glu Glu Ser Pro Pro Gly Ser Thr Lys Arg Arg Lys Ser Met Ser Gly Ala Ser Pro Lys Glu Ser Thr Asn Pro Glu Lys Ala His Glu 3S Thr Leu His Gln Ser Leu Ser Leu Ala Ser Ser Pro Lys Gly Thr Ile Glu Asn Ile Leu Asp Asn Asp Pro Arg Asp Leu Ile Gly Asp Phe Ser Lys Gly Tyr Leu Phe His Thr Val Ala Gly Lys His Gln Asp Leu Lys Tyr Ile Ser Pro Glu Ile Met Ala Ser Val Leu Asn Gly Lys Phe Ala Asn Leu Ile Lys Glu Phe Val Ile Ile Asp Cys Arg Tyr Pro Tyr Glu SS Tyr Glu Gly Gly His Ile Lys Gly Ala Val Asn Leu His Met Glu Glu ~1~3~~4- -26-Glu Val Glu Asp Phe Leu Leu Lys Lys Pro Ile Val Pro Thr Asp Gly S

Lys Arg Val Ile Val Val Phe His Cys Glu Phe Ser Ser Glu Arg Gly AGA GAT

Pro ArgMet CysArgTyr Val Glu Arg Arg Leu Gly Asn Glu Arg Asp GAG GTC

Tyr ProLys LeuHisTyr Pro Leu Tyr Leu Lys Gly Gly Tyr Glu Val CAG TGT

20 Lys GluPhe PheMetLys Cys Ser Tyr Glu Pro Pro Ser Tyr Gln Cys TTT GAC

Arg ProMet HisHisGlu Asp Lys Glu Leu Lys Lys Phe Arg Phe Asp GGG AGC

Thr LysSer ArgThrTrp Ala Glu Lys Lys Arg Glu Ile Tyr Gly Ser AGT CGTCTG AAGAAG.CTCTGAGGGCGGC 2058 AGGACCAGCC
AGCAGCAGCC

Ser ArgLeu LysLysLeu TCCATCCCCC
TTTACCCTCT

CATTTGGAGA
GGGGGCCTGG

TGGAGACCCA
GGCCATCTTG

CATTACAGAA
CTGTGCCACA

GTGGGATGAA
CCAGCCGGGG

GAGGGGACTA
GAGAAGTTTA

AG

SO (2) INFORMATION FOR SEQ ID N0:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 523 amino acids (B) TYPE: amino acid SS (D) TOPOLOGY: linear WO 94/28914 ~,16'~~ PCT/US94106365 (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID N0:2:
$ Met Glu Leu Gly Pro Ser Pro Ala Pro Arg Arg Leu Leu Phe Ala Cys Ser Pro Pro Pro Ala Ser Gln Pro Val Val Lys Ala Leu Phe Gly Ala Ser Ala Ala Gly Gly Leu Ser Pro Val Thr Asn Leu Thr Val Thr Met Asp Gln Leu Gln Gly Leu Gly Ser Asp Tyr Glu Gln Pro Leu Glu Val Lys Asn Asn Ser Asn Leu Gln Ile Met Gly Ser Ser Arg Ser Thr Asp Ser Gly Phe Cys Leu Asp Ser Pro Gly Pro Leu Asp Ser Lys Glu Asn Leu Glu Asn Pro Met Arg Arg Ile His Ser Leu Pro Gln Lys Leu Leu loo l05 llo Gly Cys Ser Pro Ala Leu Lys Arg Ser His Ser Asp Ser Leu Asp His Asp Ile Phe Gln Leu Ile Asp Pro Asp Glu Asn Lys Glu Asn Glu Ala Phe Glu Phe Lys Lys Pro Val Arg Pro Val Ser Arg Gly Cys Leu His Ser His Gly Leu Gln Glu Gly Lys Asp Leu Phe Thr Gln Arg Gln Asn Ser Ala Gln Leu Gly Met Leu Ser Ser Asn Glu Arg Asp Ser Ser Glu Pro Gly Asn Phe Ile Pro Leu Phe Thr Pro Gln Ser Pro Val Thr Ala Thr Leu Ser Asp Glu Asp Asp Gly Phe Val Asp Leu Leu Asp Gly Asp Asn Leu Lys Asn Glu Glu Glu Thr Pro Ser Cys Met Ala Ser Leu Trp Thr Ala Pro Leu Val Met Arg Thr Thr Asn Leu Asp Asn Arg Cys Lys Leu Phe Asp Ser Pro Ser Leu Cys Ser Ser Ser Thr Arg Ser Val Leu WO 94/28914 ~ PCT/LTS94106365 Lys ArgProGlu ArgSerGln GluGlu SerProPro GlySerThr Lys Arg ArgLysSer MetSerGly AlaSer ProLysGlu SerThrAsn Pro $ 290 295 300 Glu LysAlaHis GluThrLeu HisGln SerLeuSer LeuAlaSer Ser l~ Pro LysGlyThr IleGluAsn IleLeu AspAsnAsp ProArgAsp Leu Ile GlyAspPhe SerLysGly TyrLeu PheHisThr ValAlaGly Lys 1$

His GlnAsp LeuLysTyr IleSerPro GluIleMet AlaSer ValLeu 20 Asn GlyLys PheAlaAsn LeuIleLys GluPheVal IleIle AspCys Arg TyrPro TyrGluTyr GluGlyGly HisIleLys GlyAla ValAsn 2$

Leu HisMet GluGluGlu ValGluAsp PheLeuLeu LysLys ProIle Val ProThr AspGlyLys ArgValIle ValValPhe HisCys GluPhe Ser SerGlu ArgGlyPro ArgMetCys ArgTyrVal ArgGlu ArgAsp 3$ Arg LeuGly AsnGluTyr ProLysLeu HisTyrPro GluLeu TyrVal Leu LysGly GlyTyrLys GluPhePhe MetLysCys GlnSer TyrCys Glu ProPro SerTyrArg ProMetHis HisGluAsp PheLys GluAsp Leu LysLys PheArgThr LysSerArg ThrTrpAla GlyGlu LysSer 4$ 500 505 510 Lys Arg Glu Ile Tyr Ser Arg Leu Lys Lys Leu $~
(2) INFORMATION FOR SEQ ID N0:3:
(i) SEQUENCE CHARACTERISTICS:
$$ (A) LENGTH: 2886 base pairs (B) TYPE: nucleic acid WO 94!28914 PCTIUS94106365 .,..,.
_29_ z~~3~
z~
(C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 73..1773 1~
(xi) SEQID
SEQUENCE N0:3:
DESCRIPTION:

CTG CCCTGCG CCCCGCCC TC CAGCTGTG CCGGCGTTTG TTGGTCTGCC

CAGCCAGCCT
GC

ATG CAG GAG GCG G TCG
GAG C C C
GTG
C

Met !u ro ro ro ro !y er G Val Gln Glu Ala G S
P ~ P P

Ala Leu SerPro AlaGlyVal CysGlyGly AlaGln ArgProGly His Leu Pro GlyLeu LeuLeuGly SerHisGly LeuLeu GlySerPro Val Arg Ala AlaAla SerSerPro ValThrThr LeuThr GlnThrMet His 3~ 45 50 55 60 Asp Leu AlaGly LeuGlySer ArgSerArg LeuThr HisLeuSer Leu Ser Arg ArgAla SerGluSer SerLeuSer SerGlu SerSerGlu Ser Ser Asp AlaAla LeuCysMet AspSerPro SerPro LeuAspPro His Met Ala GluGln ThrPheGlu GlnAlaIle GlnAla AlaSerArg Ile Ile Arg AsnGlu GlnPheAla IleArgArg PheGln SerMetPro Val Arg Leu LeuGly HisSerPro ValLeuArg AsnIle ThrAsnSer Gln WO 94128914 ~ ~ ~ PCTIUS94106365 Ala Pro Asp Gly Arg Arg Lys Ser Glu Ala Gly Ser Gly Ala Ala Ser Ser Ser Gly Glu Asp Lys Glu Asn Asp Gly Phe Val Phe Lys Met Pro Trp AsnProThr HisPro SerSerThr HisAlaLeu AlaGluTrp Ala Ser ArgArgGlu AlaPhe AlaGlnArg ProSerSer AlaProAsp Leu Met CysLeuSer ProAsp ProLysMet GluLeuGlu GluLeuSer Pro Leu AlaLeuGly ArgPhe SerLeuThr ProAlaGlu GlyAspThr Glu Glu AspAspGly PheVal AspIleLeu GluSerAsp LeuLysAsp Asp Asp AlaValPro ProGly MetGluSer LeuIleSer AlaProLeu Val Lys ThrLeuGlu LysGlu GluGluLys AspLeuVal MetTyrSer Lys Cys GlnArgLeu PheArg SerProSer MetProCys SerValIle Arg Pro IleLeuLys ArgLeu GluArgPro GlnAspArg AspThrPro Val Gln AsnLysArg ArgArg SerValThr ProProGlu GluGlnGln Glu S0 Ala GluGluPro LysAla ArgAlaLeu ArgSerLys SerLeuCys His Asp GluIleGlu AsnLeu LeuAspSer AspHisArg GluLeuIle Gly WO 94128914 . PCTIUS94106365 6'3~~~

Asp Tyr Ser Lys Ala Phe Leu Leu Gln Thr Val Asp Gly Lys His Gln ACG

Asp LeuLysTyr IleSer ProGluThr MetValAla LeuLeu ThrGly Lys PheSerAsn IleVal AspLysPhe ValIleVal AspCys ArgTyr Pro TyrGluTyr GluGly GlyHisIle LysThrAla ValAsn LeuPro Leu GluArgAsp AlaGlu SerPheLeu LeuLysSer ProIle AlaPro Cys SerLeuAsp LysArg ValIleLeu IlePheHis CysGlu PheSer Ser GluArgGly ProArg MetCysArg PheIleArg GluArg AspArg Ala ValAsnAsp TyrPro SerLeuTyr TyrProGlu MetTyr IleLeu Lys GlyGlyTyr LysGlu PhePhePro GlnHisPro AsnPhe CysGlu Pro GlnAspTyr ArgPro MetAsnHis GluAlaPhe LysAsp GluLeu Lys ThrPheArg LeuLys ThrArgSer TrpAlaGly GluArg SerArg GCGCCAGTC

Arg GluLeuCys SerArg LeuGlnAsp Gln TGCTACCT CC CTTTC GCCAGCTGCC CTATGGGCCT CCGGGCTGA

CTTGC GAGGCCTGAA G

GGGCCTGC TG CTCAG TGGGAAAGAT GGTGTGGTGT CTGCCTGTC

GAGGC GTGCTGTCCA C

TGCC CCAGCC C TGTCATCC CATCATTTTC CATATCCTGG

CAGATTCCC TG TGCCCCCCAC

2163~2~

(2) INFORMATION
FOR SEQ
ID N0:4:

(i) SEQUENCE CHARACTERISTICS:

3S (A) LENGTH: 566 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ
ID N0:4:

Met Glu Pro Gln Pro Glu Pro Ala Pro Ser Ala Ser Pro Val Gly Leu Ala Gly Cys Gly Gly Ala Gln Arg Pro His Leu Gly Leu Val Gly Pro Leu Leu Ser His Gly Leu Leu Gly Ser Val Arg Ala Ala Gly Pro Ala Ser Ser Pro Val Thr Thr Leu Thr Gln Thr Met His Asp Leu Ala Gly SS
Leu Gly Ser Arg Ser Arg Leu Thr His Leu Ser Leu Ser Arg Arg Ala WO 94!28914 ~~ PCTlLTS94106365 .,~._ 65 70 75 g0 Ser GluSerSer LeuSer SerGluSer SerGluSer SerAsp AlaAla $

Leu CysMetAsp SerPro SerProLeu AspProHis MetAla GluGln Thr PheGluGln AlaIle GlnAlaAla SerArgIle IleArg AsnGlu Gln PheAlaIle ArgArg PheGlnSer MetProVal ArgLeu LeuGly 1$ His SerProVal LeuArg AsnIleThr AsnSerGln AlaPro AspGly Arg ArgLysSer GluAla GlySerGly AlaAlaSer SerSer GlyGlu Asp LysGluAsn AspGly PheValPhe LysMetPro TrpAsn ProThr His ProSerSer ThrHis AlaLeuAla GluTrpAla SerArg ArgGlu 2$ 195 200 205 Ala PheAlaGln ArgPro SerSerAla ProAspLeu MetCys LeuSer Pro AspProLys MetGlu LeuGluGlu LeuSerPro LeuAla LeuGly Arg PheSerLeu ThrPro AlaGluGly AspThrGlu GluAsp AspGly 3$

Phe ValAspIle LeuGlu SerAspLeu LysAspAsp AspAla ValPro Pro GlyMetGlu SerLeu IleSerAla ProLeuVal LysThr LeuGlu Lys GluGluGlu LysAsp LeuValMet TyrSerLys CysGln ArgLeu 4$ Phe ArgSerPro SerMet ProCysSer ValIleArg ProIle LeuLys Arg Leu Glu Arg Pro Gln Asp Arg Asp Thr Pro Val Gln Asn Lys Arg $0 325 330 335 Arg Arg Ser Val Thr Pro Pro Glu Glu Gln Gln Glu Ala Glu Glu Pro $$ Lys Ala Arg Ala Leu Arg Ser Lys Ser Leu Cys His Asp Glu Ile Glu WO 94/28914 ~ ~ ~ PCTIUS94/06365 Asn Leu Leu Asp Ser Asp His Arg Glu Leu Ile Gly Asp Tyr Ser Lys Ala Phe LeuLeuGln ThrValAsp GlyLysHis GlnAspLeu LysTyr Ile Ser ProGluThr MetValAla LeuLeuThr GlyLysPhe SerAsn Ile Val AspLysPhe ValIleVal AspCysArg TyrProTyr GluTyr 1$ Glu Gly GlyHisIle LysThrAla ValAsnLeu ProLeuGlu ArgAsp Ala Glu SerPheLeu LeuLysSer ProIleAla ProCysSer LeuAsp Lys Arg ValIleLeu IlePheHis CysGluPhe SerSerGlu ArgGly Pro Arg MetCysArg PheIleArg GluArgAsp ArgAlaVal AsnAsp 2$ 485 490 495 Tyr Pro SerLeuTyr TyrProGlu MetTyrIle LeuLysGly GlyTyr Lys Glu PhePhePro GlnHisPro AsnPheCys GluProGln AspTyr Arg Pro MetAsnHis GluAlaPhe LysAspGlu LeuLysThr PheArg 3$

Leu Lys ThrArgSer TrpAlaGly GluArgSer ArgArgGlu LeuCys Ser Arg LeuGlnAsp Gln 4$ (2) INFORMATION FOR SEQ ID N0:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2062 base pairs (B) TYPE: nucleic acid $0 (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: oDNA
$$
(ix) FEATURE:

.....

(A) NAME/KEY: CDS
(B) LOCATION: 211..1631 S (xi) SEQID
SEQUENCE N0:5:
DESCRIPTION:

CAGGAAGACT CTGAGTCCGA AGGCAGAGCT GCAATCTAGT

CGTTGGCCTA
CCCAGTCGGA

TAACTACCTC CTTTCCCCTA AAGTCTT CGCCTGTGTCCGA

GATTTCCTTT
CATTCTGCTC

TCTTGTAGC AGCCTCAGAC AGCTAGGTTT

TTCGAAGAC ATG TT TCA
TCT TCC
ACG
GAA
CT

Met u e Ser Ph Ser Thr Ser Glu Le Thr Arg GluGlu GlySerSer GlySerGly ProSerPhe ArgSer Asn Gln Arg LysMet LeuAsnLeu LeuLeuGlu ArgAspThr SerPhe Thr Val Cys ProAsp ValProArg ThrProVal GlyLysPhe LeuGly Asp Ser Ala AsnLeu SerIleLeu SerGlyGly ThrProLys CysCys Leu Asp Leu SerAsn LeuSerSer GlyGluIle ThrAlaThr GlnLeu Thr Thr Ser AlaAsp LeuAspGlu ThrGlyHis LeuAspSer SerGly Leu Gln Glu ValHis LeuAlaGly MetAsnHis AspGlnHis LeuMet Lys Cys Ser ProAla GlnLeuLeu CysSerThr ProAsnGly LeuAsp Arg S0 Gly His ArgLys ArgAspAla MetCysSer SerSerAla AsnLys Glu Asn Asp AsnGly AsnLeuVal AspSerGlu MetLysTyr LeuGly Ser WO 94128914 PCTlUS94106365 Pro Ile Thr Thr Val Pro Lys Leu Asp Lys Asn Pro Asn Leu Gly Glu Asp Gln Ala Glu Glu Ile Ser Asp Glu Leu Met Glu Phe Ser Leu Lys Asp Gln Glu Ala Lys Val Ser Arg Ser Gly Leu Tyr Arg Ser Pro Ser Met Pro Glu Asn Leu Asn Arg Pro Arg Leu Lys Gln Val Glu Lys Phe Lys Asp Asn Thr Ile Pro Asp Lys Val Lys Lys Lys Tyr Phe Ser Gly Gln Gly Lys Leu Arg Lys Gly Leu Cys Leu Lys Lys Thr Val Ser Leu Cys AspIle ThrIleThr GlnMetLeu GluGluAsp SerAsnGln Gly His LeuIle GlyAspPhe SerLysVal CysAlaLeu ProThrVal Ser Gly LysHis GlnAspLeu LysTyrVal AsnProGlu ThrValAla Ala Leu LeuSer GlyLysPhe GlnGlyLeu IleGluLys PheTyrVal Ile Asp Cys Arg Tyr Pro Tyr Glu Tyr Leu Gly Gly His Ile Gln Gly Ala Leu Asn Leu Tyr Ser Gln Glu Glu Leu Phe Asn Phe Phe Leu Lys Lys Pro Ile Val Pro Leu Asp Thr Gln Lys Arg Ile Ile Ile Val Phe His SS Cys Glu Phe Ser Ser Glu Arg Gly Pro Arg Met Cys Arg Cys Leu Arg ~'VO 94128914 PCTIUS94I06365 ~,3 _ ~.2~

Glu GluAspArg SerLeuAsn GlnTyr ProAlaLeu TyrTyrPro Glu S

Leu TyrIleLeu LysGlyGly TyrArg AspPhePhe ProGluTyr Met Glu LeuCysGlu ProGlnSer TyrCys ProMetHis HisGlnAsp His IS Lys ThrGluLeu LeuArgCys ArgSer GlnSerLys ValGlnGlu Gly Glu ArgGlnLeu ArgGluGln IleAla LeuLeuVal LysAspMet Ser ATAACATTCC GACACTGCAG
AGCCACTGGC
TGCTAACAAG

Pro AAAGATGTCT

(2) INFORMATION
FOR SEQ
ID N0:6:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 473 amino acids 4S (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein SO (xi) SEQUENCE DESCRIPTION: SEQ
ID N0:6:

Met Ser Glu Gly Thr Glu Ser Ser Leu Phe Gly Ser Ser Thr Arg Glu SS Ser Gly Pro Ser Phe Arg Ser Asn Gln Arg Lys Met Leu Asn Leu Leu 2163~~4 Leu Glu Arg Asp Thr Ser Phe Thr Val Cys Pro Asp Val Pro Arg Thr Pro Val Gly Lys Phe Leu Gly Asp Ser Ala Asn Leu Ser Ile Leu Ser Gly Gly Thr Pro Lys Cys Cys Leu Asp Leu Ser Asn Leu Ser Ser Gly Glu Ile Thr Ala Thr Gln Leu Thr Thr Ser Ala Asp Leu Asp Glu Thr Gly His Leu Asp Ser Ser Gly Leu Gln Glu Val His Leu Ala Gly Met Asn His Asp Gln His Leu Met Lys Cys Ser Pro Ala Gln Leu Leu Cys Ser Thr Pro Asn Gly Leu Asp Arg Gly His Arg Lys Arg Asp Ala Met Cys Ser Ser Ser Ala Asn Lys Glu Asn Asp Asn Gly Asn Leu Val Asp Ser Glu Met Lys Tyr Leu Gly Ser Pro Ile Thr Thr Val Pro Lys Leu Asp Lys Asn Pro Asn Leu Gly Glu Asp Gln Ala Glu Glu Ile Ser Asp Glu Leu Met Glu Phe Ser Leu Lys Asp Gln Glu Ala Lys Val Ser Arg Ser Gly Leu Tyr Arg Ser Pro Ser Met Pro Glu Asn Leu Asn Arg Pro Arg Leu Lys Gln Val Glu Lys Phe Lys Asp Asn Thr Ile Pro Asp Lys Val Lys Lys Lys Tyr Phe Ser Gly Gln Gly Lys Leu Arg Lys Gly Leu Cys Leu Lys Lys Thr Val Ser Leu Cys Asp Ile Thr Ile Thr Gln Met Leu Glu Glu Asp Ser Asn Gln Gly His Leu Ile Gly Asp Phe Ser Lys Val Cys Ala Leu Pro Thr Val Ser Gly Lys His Gln Asp Leu Lys Tyr Val Asn Pro Glu Thr Val Ala Ala Leu Leu Ser Gly Lys Phe Gln Gly 5$ 320 Leu Ile Glu Lys Phe Tyr Val Ile Asp Cys Arg Tyr Pro Tyr Glu Tyr 63 .~'~

Leu GlyGly HisIle GlnGlyAla LeuAsnLeu TyrSer GlnGluGlu Leu PheAsn PhePhe LeuLysLys ProIleVal ProLeu AspThrGln Lys ArgIle IleIle ValPheHis CysGluPhe SerSer GluArgGly Pro ArgMet CysArg CysLeuArg GluGluAsp ArgSer LeuAsnGln 1$ Tyr ProAla LeuTyr TyrProGlu LeuTyrIle LeuLys GlyGlyTyr Arg AspPhe PhePro GluTyrMet GluLeuCys GluPro GlnSerTyr Cys ProMet HisHis GlnAspHis LysThrGlu LeuLeu ArgCysArg 2$ Ser GlnSer LysVal GlnGluGly GluArgGln LeuArg GluGlnIle Ala LeuLeu ValLys AspMetSer Pro

Claims (36)

1. An assay method for identifying an anti-mitotic agent, comprising:

i. providing a culture of eukaryotic cells overexpressing a recombinant gene for a mitotic activator which activates a cyclin dependent kinase (CDK), wherein said mitotic activator is selected from the group consisting of cdc25 and cdc2 activating kinase (CAK); and wherein said cells have an impaired cell-cycle checkpoint caused by decreased inhibitory phosphorylation of the CDK or increased activating phosphorylation of the CDK which causes premature entry of the cells into mitosis so as to cause cell death, the premature entry into mitosis being mediated at least in part by the activation of the CDK by the mitotic activator;

ii. contacting the culture of cells with a candidate agent under conditions wherein the cell-cycle checkpoint is impaired;

iii. measuring a level of proliferation of the cells in the presence of the candidate agent; and iv. comparing the level of proliferation of the cells in the presence of the candidate agent to a level of proliferation of the cells in the absence of the candidate agent, wherein an increase in the level of proliferation in the presence of the candidate agent is indicative of anti-mitotic activity of the candidate agent.

2. The assay of claim 1, wherein the cell-cycle checkpoint comprises a G1/S
checkpoint.

3. The assay of claim 1, wherein the cell-cycle checkpoint comprises a G2/M
checkpoint.

4. The assay of claim 1, wherein the cell-cycle checkpoint is conditionally impairable.

5. The assay of claim 1, wherein the cell-cycle checkpoint impairment results in entry of the cell into mitosis before completion of replication or repair of genomic DNA of the cell.

6. The assay of claim 1, wherein the cell-cycle checkpoint impairment comprises a reduction of inhibitory phosphorylation of a cyclin dependent kinase (cdk).

7. The assay of claim 1, wherein the cell-cycle checkpoint impairment comprises a reduction of inhibitory phosphorylation of CDK.

8. The assay of claim 1, wherein the cell-cycle checkpoint impairment is induced by treatment of the cell with a hyper-mitotic agent.

9. The assay of claim 8, wherein the hyper-mitotic agent is selected from the group consisting of caffeine, 2-aminopurine, 6-dimethylaminopurine, and okadaic acid.

10. The assay of claim 1, wherein the eukaryotic cells are yeast cells.

11. The assay of claim 10, wherein the yeast cells comprise a species of the genus Schizosaccharomyces.

12. The assay of claim 1, wherein the mitotic activator is a cdc25 phosphatase.

13. The assay of claim 12, wherein the cdc25 phosphatase is a human cdc25.

14. The assay of claim 12, wherein the cdc25 phosphatase is a cdc25 of a human pathogen.

15. The assay of claim 14, wherein the cdc25 phosphatase is derived from a human pathogen which is implicated in mycotic infection.

16. The assay of claim 15, wherein the mycotic infection is a mycosis selected from the group consisting of candidiasis, aspergillosis, mucormycosis, blastomycosis, geotrichosis, cryptococcosis, chromoblastomycosis, penicilliosis, conidiosporosis, nocardiosis, coccidioidomycosis, histoplasmosis, maduromycosis, rhinosporidosis, moniliosis, para-actinomycosis, and sporotrichosis.

17. The assay of claim 15, wherein the human pathogen is selected from the group consisting of Candida albicans, Candida stellatoidea, Candida tropicalis, Candida parapsilosis, Candida krusei, Candida pseudotropicalis, Candida quillermondii, Candida rugosa, Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger, Aspergillus nidulans, Aspergillus terreus, Rhizopus arrhizus, Rhizopus oryzae, Absidia corymbifera, Absidia ramosa, and Mucor pusillus.

18. The assay of claim 14, wherein the human pathogen is Pneumocystis carinii.

19. An assay method for identifying an anti-mitotic agent, comprising i. providing a culture of eukaryotic cells having an impaired cell-cycle checkpoint caused at least in part by overexpression.of a recombinant gene encoding a mitotic activator selected from the group consisting of cdc25 and CAK, wherein said mitotic activator activates a cyclin dependent kinase (CDK) thereby causing premature entry of the cells into mitosis so as to cause cell death;

ii. contacting the culture of cells with a candidate agent under conditions wherein the cell-cycle checkpoint is impaired;

iii. measuring a level of proliferation of the cells in the presence of the candidate agent; and iv. comparing the level of proliferation of the cells in the presence of the candidate agent to a level of proliferation of the cells in the absence of the candidate agent, wherein an increase in the level of proliferation in the presence of the candidate agent is indicative of anti-mitotic activity of the candidate agent.

20. The assay of claim 19, wherein the cell-cycle checkpoint comprises a G1/S
checkpoint.

21. The assay of claim 19, wherein the cell-cycle checkpoint comprises a G2/M
checkpoint.

22. The assay of claim 19, wherein the cell-cycle checkpoint is conditionally impairable.

23. An assay method for identifying an inhibitor of a cdc25 phosphatase, comprising i. providing a culture of Schizosaccharomyces cells having a conditionally impairable weel protein kinase which, when impaired, causes inhibition of proliferation of the Schizosaccharomyces cells by facilitating premature entry of the Schizosaccharomyces cells into mitosis, the premature entry into mitosis being mediated at least in part by a cdc25 phosphatase and a reduced level of inhibitory phosphorylation of a cdc2 protein kinase by the weel protein kinase, wherein the impairment of the weel protein kinase activity is caused by overexpression of a niml activator in the Schizosaccharomyces cell;

ii. contacting the culture of Schizosaccharomyces cells with a test compound under conditions wherein the wee 1 kinase is impaired;

iii. measuring a level of proliferation of the Schizosaccharomyces cells in the presence of the test compound; and iv. comparing the level of proliferation of the Schizosaccharomyces cells in the presence of the test compound to a level of proliferation of the Schizosaccharomyces cells in the absence of the test compound, wherein an increase in the level of proliferation in the presence of the test compound is indicative of inhibition of the cdc25 phosphatase by the test compound.

24. The assay of claim 23, wherein the Schizosaccharomyces cell is an Schizosaccharomyces pombe cell.

25. The assay of claim 23, wherein the Schizosaccharomyces cell is a conditional wee phenotype.

26. The assay of claim 25, wherein the Schizosaccharomyces cell is a weel -SO
mutant.

27. The assay of claim 23, wherein the Schizosaccharomyces cell is an OP-niml mutant.

28. The assay of claim 23, wherein the cdc25 phosphatase activity is a recombinant gene product expressed in the Schizosaccharomyces cell, and the Schizosaccharomyces cell lacks a functional endogenous cdc25 phosphatase activity.

29. The assay of claim 28, wherein the cdc25 phosphatase activity is a human cdc25 or homolog thereof.

30. The assay of claim 29, wherein the human cdc25 is selected from a group consisting of cdc25A, cdc25B and cdc25C.

31. The assay of claim 28, wherein the cdc25 phosphatase activity is a human pathogen cdc25 or homolog thereof.

32. The assay of claim 29, wherein the human pathogen is a fungus implicated in a mycotic infection selected from a group consisting of candidiasis, aspergillosis, mucormycosis, blastomycosis, geotrichosis, cryptococcosis, chromoblastomycosis, coccidioidomycosis, conidiosporosis, histoplasmosis, maduromycosis, rhinosporidosis, nocardiosis, para-actinomycosis, penicilliosis, moniliosis, and sporotrichosis.

33. The assay of claim 1 or 19, wherein the mitotic activator is a cdc2 activating kinase (CAK).

34. An assay method for identifying an anti-mitotic agent, comprising i. providing a culture of eukaryotic cells having an impaired G1/S cell-cycle checkpoint caused by either decreased inhibitory phosphorylation of a cyclin dependent kinase (CDK), or by increased activating phosphorylation of the CDK, which impairment causes premature entry of the cells into mitosis so as to cause cell death, wherein said cell-cycle checkpoint impairment comprises impaired weel protein kinase activity, impaired mild protein kinase activity or over expression of a niml gene product;

ii. contacting the culture of cells with a candidate agent under conditions wherein the cell-cycle checkpoint is impaired;

iii. measuring a level of proliferation of the cells in the presence of the candidate agent; and iv. comparing the level of proliferation of the cells in the presence of the candidate agent to a level of proliferation of the cells in the absence of the candidate agent, wherein an increase in the level of proliferation in the presence of the candidate agent is indicative of anti-mitotic activity of the candidate agent.

35. The assay of claim 1, 28 or 34, wherein the eukaryotic cells are mammalian cells.

36. The assay of claim 35, wherein the mammalian cells are human cells.