WO2007120463A2 - Compositions and methods for modulation of nek2 kinase activity - Google Patents

Abstract

Described are serine and threonine-containing peptides derived from the proteins c-NAPl and NEK2 that constitute phosphorylation substrates for the serine/threonine kinase NEK2. These peptides can be used to measure kinase activity of a NEK2 protein and to identify compounds that modulate phosphorylation of a NEK2 substrate by a NEK2 protein. The peptides can also be used as competitive inhibitors of NEK2 kinase activity.

Description

COMPOSITIONS AND METHODS FOR MODULATION OF NEK2 KINASE ACTIVITY

Technical Field The invention relates to protein chemistry and cellular and molecular biology.

Background

Mitotic division is a highly ordered and regulated process orchestrated by a number of specialized organelles and protein structures. One of the structures that must be assembled properly is the mitotic spindle, whose primary function is to separate the duplicated chromosomes to their respective daughter cells. The source of these mitotic spindles are the centrosomes that duplicate at the on-set of S-phase, migrate to opposite poles, and begin to emanate α/β microtubule heterodimers that ultimately form the spindle. Central to this separation event is a serine/threonine kinase termed Nek2 (Never in Mitosis Arrest a Related Kinase 2) that phosphorylates a large linker protein termed c-NAPl (centrosomal-iVek2 Associated Protein /) (Fry et al. (2002) Oncogene 21 :6184- 94; and Fry et al. (1998) J Cell Biol. 141:1563-74). This phosphorylation event triggers the ubiquitin-mediated degradation of c-NAPl, thereby releasing the duplicated centrosomes and subsequently permitting them to migrate (Mayor et al. (2000) J. Cell Biol. 151:837-46; and Mayor et al. (2002) J. Cell Sci 115:3275-84).

Nek2 is the mammalian homolog of the NIMA protein in filamentous fungus Aspergillus nidulans that has been shown to be critical for mitotic entry (Fry et al. (1995) J. Biol. Chem. 270:12899-905). While there are nine Nek family members, Nek2A and Nek2B, which are alternative splice variants, are the only family members selectively up- regulated during G2/M and are localized primarily in the centrosomes of rapidly proliferating cells (Schultz et al. (1994) Cell Growth & Differentiation 5: 625-635; and Fry et al. (2002) Oncogene 21:6184-94). High expression of Nek2 compared with normal tissue has been observed in lung, colon, breast, and prostate carcinomas as well as B-cell lymphomas (Hayward et al. (2004) Cancer Research 64:7370-76; and de Vos et al. (2003) Laboratory Investigation 83:271-85). Summary

The invention is based, at least in part, on the discovery that selected serine or threonine-containing peptides derived from the proteins c-NAPl and NEK2 constitute phosphorylation substrates for the serine/threonine kinase NEK2. These peptides can be used in screening assays to identify compounds that modulate phosphorylation of a NEK2 substrate by a NEK2 protein. Compounds that inhibit NEK2 kinase activity are expected to interfere with centrosome separation and result in decreased cellular proliferation.

Described herein is an isolated peptide that contains the amino acid sequence of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO: 13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO: 17, or a variant of any of the foregoing SEQ ID NOS, wherein the variant is a phosphorylation substrate of NEK2.

In some embodiments, the peptide is less than 100 amino acids in length. For example, the peptide can be less than 75, 50, 40, 30, or 25 amino acids in length. In some embodiments, the peptide is 20 or fewer or 15 or fewer amino acids in length.

In some embodiments, the peptide contains a variant of any one of SEQ ID NOS: 1-17 in which at least one but not more than five amino acid residues are substituted, deleted, or inserted, wherein the variant is a phosphorylation substrate of NEK2. For example, a variant can be such that at least one but not more than two amino acid residues of any one of SEQ ID NOS: 1-17 are substituted, deleted, or inserted. Alternatively, a variant can be such that only one amino acid residue of any one of SEQ ID NOS: 1-17 is substituted, deleted, or inserted.

In some embodiments, the peptide contains the amino fccid sequence of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ED NO: 14, SEQ ID NO:15, SEQ ID NO:16, or SEQ ID NO: 17.

In some embodiments, the peptide consists of the amino acid sequence of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, or SEQ ID NO: 17.

Peptides described herein can optionally contain a heterologous amino acid sequence.

Peptides described herein can optionally be phosphorylated on a serine or threonine residue.

Peptides described herein can optionally be conjugated to a first member of a specific binding pair (e.g., biotin or streptavidin).

Peptides described herein can optionally be conjugated to a detection moiety (e.g., a fluorescent agent such a europium, terbium, green-fluorescent protein).

Also described herein is an isolated antibody that specifically binds to a peptide whose amino acid sequence consists of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO: 14, SEQ ID NO:15, SEQ ID NO:16, or SEQ ID NO:17. In some embodiments, the antibody preferentially binds the peptide when phosphorylated on a serine or threonine amino acid residue.

Also described herein is a method of generating an immune response in a mammal by administering to the mammal an effective amount of a peptide described herein.

Also described herein is a method of detecting kinase activity of a NEK2 protein by: (i) contacting a NEK2 protein with a peptide described herein under conditions effective to permit phosphorylation of the peptide; and (ii) measuring phosphorylation of the peptide, wherein phosphorylation of the peptide indicates kinase activity of the NEK2 protein.

Also described herein is a method of identifying a compound that inhibits phosphorylation of aNEK2 substrate by: (i) contacting, in the presence of a candidate compound, a NEK2 protein with a peptide described herein; and (ii) measuring phosphorylation of the peptide, wherein decreased phosphorylation of the peptide in the presence of the candidate compound as compared to phosphorylation of the peptide that occurs in the absence of the candidate compound indicates that the candidate compound inhibits phosphorylation of a NEK2 substrate by the NEK2 protein.

In some embodiments of the foregoing methods, measuring phosphorylation of the peptide includes: (i) contacting the peptide with an antibody that (a) is conjugated to a first fluorescent agent and (b) specifically binds to the peptide when the peptide is phosphorylated on a serine or threonine residue, wherein the peptide is conjugated to a second fluorescent agent; and (ii) detecting the occurrence of fluorescence resonance energy transfer between the first fluorescent agent and the second fluorescent agent as an indicator of phosphorylation of the peptide.

In some embodiments of the foregoing methods, measuring phosphorylation of the peptide includes: (i) contacting the peptide with an antibody that (a) is conjugated to a detection moiety and (b) specifically binds to the peptide when the peptide is phosphorylated on a serine or threonine residue; removing antibody that is not bound to the peptide; and (ii) detecting the detection moiety associated with the peptide as an indicator of phosphorylation of the peptide.

In some embodiments of the foregoing methods, measuring phosphorylation of the peptide includes passaging the peptide through a stationary phase, wherein retardation of the peptide during passage through the stationary phase indicates the phosphorylation status of the peptide.

Also described herein is a method of inhibiting phosphorylation of a NEK2 substrate by contacting a NEK2 protein with a composition that that binds to the NEK2 protein and inhibits phosphorylation of a NEK2 substrate by the NEK2 protein. The composition can optionally be a peptide (e.g., a peptide described herein).

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the exemplary methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present application, including definitions, will control. The materials, methods, and examples are illustrative only and not intended to be limiting.

Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.

Detailed Description

The peptides described herein can be used to identify compounds that modulate phosphorylation of a NEK2 substrate and/or can be used as competitive inhibitors of NEK2 kinase activity. NEK2 is localized primarily in the centrosomes of rapidly proliferating cells and is expressed at high levels in certain cancers. As a result, compounds that inhibit NEK2 phosphorylation are expected to interfere with centrosome separation and decrease cellular proliferation.

NEK2 Peptide Substrates

As detailed in the accompanying Examples, the c-NAPl andNEK2 peptides of SEQ ID NOS:1-17 (see Tables 1 and 2) are phosphorylation substrates for the kinase NEK2. Described herein are isolated peptides that contain the amino acid sequence of any one of SEQ ID NOS: 1-17 or a biologically active variant thereof.

As used herein, an isolated peptide is a peptide that is separated from those components (proteins and other naturally-occurring organic molecules) that naturally accompany it. Typically, a peptide is isolated when it constitutes at least 60%, by weight, of the protein in a preparation. In some embodiments, the peptide in the preparation consists of at least 75%, at least 90%, or at least 99%, by weight, of the protein in a preparation.

As used herein, a biologically active variant of any one of SEQ ID NOS: 1-17 is a peptide that retains the ability to function as a phosphorylation substrate for the kinase NEK2. Numerous assays are described herein that allow for a determination of whether a peptide functions as a phosphorylation substrate for NEK2.

In some embodiments, a biologically active variant of any one of SEQ ID NOS: 1- 17 has at least one but not more than five amino acids substituted, deleted, or inserted (as compared to the amino acid sequence of SEQ ID NOS: 1-17). In some biologically active variants, not more than four, three, two, or one amino acids are substituted, deleted, or inserted. Substitutions or deletions can be made at amino acid residues within SEQ ID NOS: 1-17 other than serine or threonine residues that are the target of phosphorylation by NEK2.

In some embodiments, a biologically active variant is prepared by means of conservative substitution at one or more amino acid residues within any one of SEQ ID NOS: 1-17. A conservative substitution is the substitution of one amino acid for another with similar characteristics. Conservative substitutions include substitutions within the following groups: valine, alanine and glycine; leucine, valine, and isoleucine; aspartic acid and glutamic acid; asparagine and glutamine; serine, cysteine, and threonine; lysine and arginine; and phenylalanine and tyrosine. The non-polar hydrophobic amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan and methionine. The polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine and glutamine. The positively charged (basic) amino acids include arginine, lysine and histidine. The negatively charged (acidic) amino acids include aspartic acid and glutamic acid. Any substitution of one member of the above-mentioned polar, basic or acidic groups by another member of the same group can be deemed a conservative substitution.

In some embodiments, a peptide containing any one of SEQ ID NOS: 1-17 or a. biologically active variant of any one of SEQ ID NOS:1-17 is synthesized such that it is phosphorylated on a serine or threonine residue (i.e., a serine or threonine residue that is present in any one of SEQ ID NOS: 1-17). In some instances, a peptide described herein is modified to substitute a serine or threonine residue of any one of SEQ ID NOS: 1-17 with an alternative amino acid (e.g., alanine) so as to create a peptide that cannot be phosphorylated by NEK2 (the modified peptide can be used, e.g., as a control in certain screening assays described herein).

A peptide containing the amino acid sequence of any one of SEQ ID NOS: 1-17 or a biologically active variant thereof can vary in length. For example, the peptide can contain the amino acid sequence of any one of SEQ ID NOS: 1 -17 (or a biologically active variant of any one of SEQ ID NOS: 1-17) as well as additional amino acid sequences added to the carboxy and/or amino termini. As detailed in the accompanying Examples, the addition of amino acids to the amino terminus of c-NAPl peptide #97 (SEQ ID NO: 6) yielded a peptide that functions as a NEK2 phosphorylation substrate. In those instances in which amino acids are added to the carboxy and/or amino termini, the resulting peptide can optionally be less than 100, less than 75, less than 50, less than 40, less than 30, or less than 25 amino acids in length. In some embodiments, the resulting peptide is 20 or fewer or 15 or fewer amino acids in length.

Peptides can be synthesized chemically using standard peptide synthesis techniques. See, e.g., Stewart, et al., Solid Phase Peptide Synthesis (2d ed., 1984). Peptides can also be produced by recombinant DNA techniques. For example, a nucleic acid molecule encoding a peptide can be inserted into a vector (e.g., an expression vector) and the nucleic acid can be introduced into a cell. Site-directed mutagenesis can optionally be used, in which a specific nucleotide (or, if desired a small number of specific nucleotides) is changed in order to change a single amino acid (or, if desired, a small number of amino acid residues) in the encoded peptide. Suitable cells for expression of the peptide include, e.g., mammalian cells (such as human cells or CHO cells), fungal cells, yeast cells, insect cells, and bacterial cells (e.g., E. coli). When expressed in a recombinant cell, the cell is cultured under conditions allowing for expression of the peptide. The peptide can optionally be recovered from a cell suspension.

A fusion protein can be prepared that contains the amino acid sequence of any one SEQ ID NOS: 1-17 (or a biologically active variant of any one of SEQ ID NOS: 1-17) and heterologous amino acid sequences. Heterologous, as used herein when referring to an amino acid sequence, refers to a sequence that originates from a source other than the naturally occurring polypeptide from which the peptide is derived. A fusion protein containing a peptide described herein and a heterologous amino acid sequence thus does not correspond in sequence to all or part of a naturally occurring protein. A heterologous sequence can be, for example a sequence used for purification of the recombinant peptide (e.g., GST sequences or a histidine tag). In another embodiment, the fusion protein contains a signal sequence from another protein. In certain host cells (e.g., mammalian host cells), expression and/or secretion of the peptide can be increased through use of a heterologous signal sequence. Screening Assays

Described herein are methods for identifying a candidate compound that modulates (inhibits or stimulates) phosphorylation of a NEK2 substrate (e.g., the peptide of any one of SEQ ID NOS: 1-17 or a biologically active variant thereof) by a NEK2 protein.

Examples of cell free assay conditions in which NEK2 kinase activity (i.e., the ability of a NEK2 protein to phosphorylate a NEK2 substrate) can be measured are described in detail in the accompanying Examples. Generally, reactions involve the • addition of a NEK2 protein and a NEK2 substrate (e.g., the peptide of any one of SEQ ID NOS: 1-17 or a biologically active variant thereof) in the presence of ATP and magnesium (e.g., MgCl or MgOAc) or manganese, in a pH-buffered, suitable aqueous medium (e.g., Tris-buffered saline or HEPES), at physiologic temperature (e.g., 37°C) for a suitable amount of time (e.g., 30 minutes, 60 minutes, or 120 minutes). Kinase reaction conditions and general reaction optimization methodologies are well known in the art. The NEK2 protein can be, e.g., purified, recombinant enzyme (e.g., recombinantly expressed in a bacterial cell, a yeast cell, an insect cell, or a mammalian cell) or can be isolated from a naturally NEK2 -expressing host (e.g., a mammalian cell line such as a human cell line).

Human NEK2A (GenBank™ Reference NP_002488) is 445 amino acids in length and has the following amino acid sequence: MPSRAED YEVL YTIGTGSYGRCQKIRR

KSDGKJLVWKELDYGSMTEAEKQMLVSEVNLLRELKHPNΓVRYYDRIIDRTNTT LYIVMEYCEGGDLASVITKGTKERQYLDEEFVLRVMTQLTLALKECHRRSDGGH TVLHRDLKPANVFLDGKQNVKLGDFGLARILNHDTSFAKTFVGTPYYMSPEQM NRMSYNEKSDIWSLGCLLYELCALMPPFTAFSQKELAGKIREGKFRRIP YRYSDE LNEIITRMLNLKDYHRPSVEEILENPLIADLVADEQRRNLERRGRQLGEPEKSQDS SPVLSELKLKEIQLQERERALKAREERLEQKEQELCVRERLAEDKLARAENLLKN YSLLKERKFLSLASNPELLNLPSSVIKKKVHFSGESKENIMRSENSESQLTSKSKC KDLKKRLHAAQLRAQALSDIEKNYQLKSRQILGMR (SEQ ID NO: 19).

Human NEK2B (GenBank™ Reference AAK92212) is 384 amino acids in length and has the following amino acid sequence: MPSRAED YEVLYTIGTGSYGRCQKIRR KSDGKILVWKELDYGSMTEAEKQMLVSEVNLLRELKHPNIVRYYDRIIDRTNTT

LYIVMEYCEGGDLASVΓΓKGTKERQYLDEEFVLRVMTQLTLALKECHRRSDGGH

TVLHRDLKPANVFLDGKQNVKLGDFGLARILNHDTSFAKTFVGTPYYMSPEQM

NRMSYNEKSDIWSLGCLLYELCALMPPFTAFSQKELAGKIREGKFRRIP YRYSDE

LNEIITRMLNLKDYHRPSVEEILENPLIADLVADEQRRNLERRGRQLGEPEKSQDS

SPVLSELKLKEIQLQERERALKAREERLEQKEQELCVRERLAEDKLARAENLLKN

YSLLKERKFLSLASNPGMRINLVNRSWCYK (SEQ ID NO:20).

The kinase domain of human NEK2A and NEK2B is contained within amino acid residues 1-271 of each of SEQ ID NO: 19 and SEQ ID NO:20 (Fry et al. (2002) Oncogene 21:6184-94).

ANEK2 protein used in the methods described herein contains the sequence of a naturally occurring NEK2 polypeptide or a fragment or variant thereof that retains serine/threonine kinase activity. A variant NEK2 polypeptide can contain one or more additions, substitutions, and/or deletions relative to the sequence of a naturally occurring NEK2 polypeptide.

In some embodiments, a variant NEK2 polypeptide (i) contains one or more amino acid substitutions, and (ii) is at least 70%, 80%, 85%, 90%, 95%, 98% or 99% identical to SEQ ID NO: 19 or SEQ ID NO:20 (or 70%, 80%, 85%, 90%, 95%, 98% or 99% identical to amino acids 1-271 of SEQ ID NO: 19 or SEQ ID NO:20). A variant NEK2 polypeptide differing in sequence from SEQ ID NO: 19 or SEQ ID NO:20 (or differing in sequence from amino acids 1-271 of SEQ ID NO: 19 or SEQ ID NO:20) may include one or more amino acid substitutions (conservative or non-conservative), one or more deletions, and/or one or more insertions. In an exemplary embodiment, a biologically active NEK2 fragment contains amino acid residues 1-271 of SEQ ID NO: 19 and SEQ ID NO:20.

The ability of a candidate compound to modulate phosphorylation of a NEK2 substrate (e.g., the peptide of any one of SEQ ID NOS: 1-17 or a biologically active variant thereof) by a NEK2 protein can be directly measured by adding to a kinase reaction a source of ATP comprising an ATP linked to a detectably-labeled gamma- phosphate moiety. The detectable label can be, for example, a radioisotope label (e.g., ³³P or ³²P). The ability of a candidate compound to modulate phosphorylation of a NEK2 substrate by a NEK2 protein can be measured by detecting the amount of labeled gamma- phosphate incorporated into the substrate in the presence or absence of the candidate compound. Determining the amount of the labeled phospho-substrate can be accomplished through the use of instrumentation that detects or quantitates radioisotope decay or appropriate autoradiographic film.

The ability of a candidate compound to modulate phosphorylation of a NEK2 substrate (e.g., the peptide of any one of SEQ ID NOS:1-17 or a biologically active variant thereof) by a NEK2 protein can also be determined by analyzing the rate of the substrate's physical passage through a stationary phase matrix (e.g., HPLC or TLC methodology). Following a kinase reaction described herein, samples can be resuspended in an appropriate solvent (or liquid phase) and actively or passively passaged over a stationary phase matrix (e.g., a silica-based gel or plate), which can retard (i.e., increase the retention time of) a modified substrate on the basis of physical properties (e.g., size, hydrophobicity, or charge). The occurrence or non-occurrence of phosphorylation of the NEK2 substrate by theNEK2 protein can be determined by measuring the retention time between the passage of a phosphorylated peptide compared to a non-phosphorylated peptide over the stationary phase matrix. For more details about HPLC methodology, see, for example, Nageswara-Rao et al. J. Pharm. Biomed. Anal. 2003 Oct 15;33(3):335-77). Alternatively, following the kinase reaction step of the procedure, the mixture can be resuspended in buffer and subjected to polyacrylamide gel electrophoresis (PAGE). PAGE-resolved proteins, separated by size, can then be transferred to a filter membrane (e.g., nitrocellulose) and subjected to western blot techniques using antibodies specific to, e.g., the NEK2 substrate. The extent of phosphorylation of a NEK2 substrate, in the presence or absence of the candidate compound, can be detected by comparing the relative position of the phosphorylated species of substrate with the non-phosphorylated species of substrate.

The ability of a candidate compound to modulate phosphorylation of a NEK2 substrate (e.g., the peptide of any one of SEQ ID NOS:1-17 or a biologically active variant thereof) by a NEK2 protein can also be determined by immunoassay. A NEK2 kinase reaction can performed in the presence of a NEK2 substrate as detailed herein, followed by addition of a detection antibody that specifically recognizes a phosphorylated residue in a NEK2 substrate. The extent of phosphorylation of a NEK2 substrate in the presence of the candidate compound can be determined by comparing the amount of antibody bound to the NEK2 substrate (following the kinase reaction) as compared the amount of antibody bound to a control substrate (e.g., a substrate not exposed to the candidate compound and/or the NEK2 protein).

For the purposes of detection, an immunoassay can be performed with an antibody that bears a detection moiety (e.g., a fluorescent agent such a europium, terbium, green-fluorescent protein, or a fluorescent dye). The NEK2 substrate can be conjugated directly to a solid-phase matrix (e.g., a multiwell assay plate, nitrocellulose, agarose, sepharose, or magnetic beads) or it can be conjugated to a first member of a specific binding pair (e.g., biotin or streptavidin) that attaches to a solid-phase matrix upon binding to a second member of the specific binding pair (e.g., streptavidin or biotin). Such attachment to a solid-phase matrix allows the NEK2 substrate to be purified away from reaction components prior to contact with the detection antibody and also allows for subsequent washing of unbound antibody. An example of an immunoassay detection method that can be used to identify a candidate compound that modulates phosphorylation of aNEK2 substrate by a NEK2 protein is the commercially available DELFIA® system of Perkin Elmer Life Sciences (Emeryville, CA).

An immunoassay method can alternatively use two detection moieties in fluorescence resonance energy transfer (FRET), which entails the radiationless transfer of energy from a donor molecule to an acceptor molecule. The donor molecule can be a dye or chromophore that initially absorbs energy and the acceptor can be a chromophore to which the energy is subsequently transferred (called a donor/acceptor pair). This resonance interaction occurs over greater than inter-atomic distances, without conversion to thermal energy and without any molecular collision. Due to its sensitivity to distance, FRET is extremely useful in investigating protein-protein interactions and enzymatic reactions.

In one example of a FRET method, a NEK2 substrate (e.g., the peptide of any one of SEQ ID NOS: 1-17 or a biologically active variant thereof) is conjugated to an energy acceptor molecule and an anti-phospho-serine/threonine antibody is conjugated to an energy donor molecule. Alternatively, theNEK2 substrate can be conjugated to the energy donor molecule and the anti-phospho-serine/threonine antibody can be conjugated to the energy acceptor molecule. The NEK2 substrate can be bound directly to either the FRET energy acceptor or donor or can be conjugated to a first member of a specific binding pair (e.g., biotin) with the FRET energy acceptor or donor being conjugated to a second member of the specific binding pair (e.g., streptavidin). Phosphorylation of the NEK2 substrate by a NEK2 protein can be determined by measuring the amount of FRET following a kinase reaction performed in the presence or absence of a candidate compound. An example of a FRET method that can be used to identify a candidate compound that modulates phosphorylation of a NEK2 substrate by a NEK2 protein is the commercially available LANCE™ assay of Perkin Elmer Life Sciences (Emeryville, CA).

Candidate compounds that can be used in the methods described herein include various chemical classes and include small organic molecules having a molecular weight in the range of, e.g., 50 to 2,500 daltons. Candidate compounds can optionally contain functional groups that promote interaction with proteins (e.g., hydrogen bonding) and can include at least an amine, carbonyl, hydroxyl, or carboxyl group (or at least two of the functional chemical groups). Candidate compounds can optionally contain cyclical carbon or heterocyclic structures and/or aromatic or polyaromatic structures (e.g., purine core) substituted with one or more of the above functional groups.

Candidate compounds can also include biomolecules including, but not limited to, peptides, polypeptides, proteins, antibodies, peptidomimetics, saccharides, fatty acids, steroids, purines, pyrimidines, derivatives or structural analogues thereof.

Candidate compounds can be identified from a number of potential sources, including chemical libraries, natural product libraries, and combinatorial libraries comprised of random peptides, oligonucleotides, or organic molecules. Chemical libraries can consist of random chemical structures, some of which are analogs of known compounds or analogs or compounds that have been identified as hits or leads in other drug discovery screens, while others are derived from natural products, and still others arise from non-directed synthetic organic chemistry. Natural product libraries can include polyketides, non-ribosomal peptides, and variants (non-naturally occurring) thereof. For a review, see Science 282:63-68 (1998). Combinatorial libraries can be composed or large numbers of peptides, oligonucleotides, or organic compounds as a mixture.

Peptide libraries can be prepared by traditional automated synthesis methods or by use of recombinant nucleic acids. Libraries of interest include peptide combinatorial, protein, peptidomimetic, multiparallel synthetic collection, recombinatorial, and polypeptide libraries. For a review of combinatorial chemistry and libraries created therefrom, see Myers, Curr. Opin. Biotechnol. 8:701-707 (1997). Identification of candidate compounds through the use of the various libraries permits subsequent modification of the candidate compound hit or lead to optimize the capacity of the hit or lead to modulate phosphorylation of a NEK2 substrate by a NEK2 protein.

Candidate compounds identified herein can be synthesized by any chemical or biological method. The candidate compounds can also be pure, or may be in a heterologous composition, and can be prepared in an assay-, physiologic-, or pharmaceutically- acceptable diluent or carrier. This composition can also contain additional compounds or constituents that do not bind to or modulate the kinase activity of a NEK2 protein.

Screening assays can optionally be performed in formats that allow for rapid preparation, processing, and analysis of multiple reactions. This can be, for example, in multi-well assay plates (e.g., 96 wells or 386 wells). Stock solutions for various agents can be provided manually or robotically, and subsequent pipetting, diluting, mixing, distribution, washing, incubating, sample readout, data collection and/or analysis can be done robotically using commercially available analysis software, robotics, and detection instrumentation capable of detecting the signal generated from the assay. Examples of such detectors include, but are not limited to, spectrophotometers, luminometers, fluorimeters, and devices that measure radioisotope decay.

The ability of a candidate compound (e.g., a compound identified as a NEK2 kinase inhibitor by a cell free assay described herein) to modulate NEK2 kinase activity can also be evaluated in a cell-based assay. The effects of a candidate compound on the biological activity of NEK2 can be measured, for example, by monitoring the phosphorylation state of an endogenous NEK2 substrate (e.g., PPl or c-NAPl). The phosphorylation state of the substrate can be measured in intact cells using antibody- mediated immunofluorescence or immunohistochemical techniques. The phosphorylation state of a endogenous substrate can alternatively be measured, for example, by solubilizing cells and subjecting the solubilized extracts to PAGE, followed by western blotting with antibodies specific for phosphorylated residues in the NEK2 substrate. Alternatively, an antibody that recognizes a non-phosphorylated NEK2 substrate can also be used to detect changes in protein mobility consistent with protein modification (e.g., phosphorylation).

Methods of Generating Antibodies and Inducing an Immune Response

A peptide described herein (e.g., the peptide of any one of SEQ ID NOS: 1-17 or a biologically active variant thereof) can be used as an immunogen to generate antibodies that bind to the peptide. The peptide used as an immunogen contains an epitope present in any one of SEQ ED NOS: 1-17 such that an antibody raised against the peptide is capable of forming a specific immune complex with any one of SEQ DD NOS:1-17. In some embodiments, the peptide is phosphorylated on a serine or threonine residue that is present in any one of SEQ ID NOS:1-17. As a result, antibodies can be generated that preferentially bind to the peptide when phosphorylated on a serine or threonine amino acid residue.

A peptide can be used to prepare antibodies by immunizing a suitable subject, (e.g., rabbit, goat, mouse, or other mammal) with the peptide. An appropriate immunogenic preparation can contain, for example, a chemically synthesized peptide or a recombinantly expressed peptide. The preparation can further include an adjuvant, such as Freund's complete or incomplete adjuvant, or similar immunostimulatory agent. Immunization of a suitable subject with an immunogenic peptide preparation induces a polyclonal anti-peptide antibody response.

The term antibody as used herein refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules (i.e., molecules that contain an antigen binding site that specifically bind to the peptide). An antibody that specifically binds to a peptide described herein is an antibody that binds the peptide, but does not substantially bind other molecules in a sample. Examples of immunologically active portions of immunoglobulin molecules include F(ab) and F(ab')2 fragments. The anti-peptide antibody can be a monoclonal antibody or a preparation of polyclonal antibodies. The term monoclonal antibody, as used herein, refers to a population of antibody molecules that contain only one species of an antigen binding site capable of immunoreacting with the peptide. A monoclonal antibody composition thus typically displays a single binding affinity for a particular peptide with which it immunoreacts.

Polyclonal anti-peptide antibodies can be prepared as described above by immunizing a suitable subject with a peptide immunogen. The anti-peptide antibody titer in the immunized subject can be monitored over time by standard techniques, such as with an enzyme linked immunosorbent assay (ELISA) using immobilized peptide. If desired, the antibody molecules directed against the peptide can be isolated from the mammal (e.g., from the blood) and further purified by techniques such as protein A chromatography to obtain the IgG fraction. At an appropriate time after immunization, e.g., when the anti-peptide antibody titers are highest, antibody-producing cells can be obtained from the subject and used to prepare monoclonal antibodies by standard techniques, such as the hybridoma technique originally described by Kohler and Milstein (1975) Nature 256:495-497, the human B cell hybridoma technique (Kozbor et al. (1983) Immunol Today 4:72), or the EBV-hybridoma technique (Cole et al. (1985), Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96). Any of the many well known protocols used for fusing lymphocytes and immortalized cell lines can be applied for the purpose of generating an anti-peptide monoclonal antibody (see, e.g., Current Protocols in Immunology, supra; Galfre et al. (1977) Nature 266:55052; R.H. Kenneth, in Monoclonal Antibodies: A New Dimension In Biological Analyses, Plenum Publishing Corp., New York, New York (1980); and Lerner (1981) Yale J. Biol. Med., 54:387-402).

As an alternative to preparing monoclonal antibody-secreting hybridomas, a monoclonal anti-peptide antibody can be identified and isolated by screening a recombinant combinatorial immunoglobulin library (e.g., an antibody phage display library) with a peptide described herein to isolate immunoglobulin library members that bind the peptide. An anti-peptide antibody (e.g., a monoclonal antibody) can be used to isolate the peptide by techniques such as affinity chromatography or immunoprecipitation. Moreover, an anti- peptide antibody can be used to detect the peptide in screening assays described herein. An antibody can optionally be coupled to a detectable substance such as an enzyme (e.g., horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase), a prosthetic group (e.g., streptavidin/biotin or avidin/biotin), a fluorescent material (e.g., umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, or phycoerythrin), a luminescent material (e.g., luminal), a bioluminescent material (e.g., luciferase, luciferin, or aequorin), or a radioactive materials (e.g., ¹²⁵1, ¹³¹I, ³⁵S, or ³H).

Methods of Inhibiting Phosphorylation of aNek2 Substrate

A compound identified by a method described herein can be used to inhibit phosphorylation of a NEK2 substrate by a NEK2 protein. Similarly, peptides that contain the amino acid sequence of any one of SEQ ID NOS:1-17 (or a biologically active variant thereof) can be used as competitive inhibitors of NEK2 kinase activity.

Methods of inhibiting phosphorylation of a NEK2 substrate can be performed in vitro (e.g., by culturing a cell with the compound) or in vivo (e.g., by administering the compound to a subject). In these methods, the compound inhibits the ability of NEK2 to phosphorylate an endogenous, physiological substrate. Inhibition of NEK2 kinase activity is expected to interfere with centrosome separation and result in decreased cellular proliferation.

Examples of peptides that can be used as competitive inhibitors of NEK2 kinase activity include the NEK2 peptide substrates described herein, such as a peptide that is less than 50 amino acids in length and contains: (i) SEQ ID NO: 1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9^' SEQ ID NOrIO, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, or SEQ ID NO: 17; or (ii) a variant of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8₅ SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 11 , SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, or SEQ ID NO: 17 in which at least one but not more than five amino acid residues are substituted, deleted, or inserted, wherein the variant is a phosphorylation substrate of NEK2. Methods of facilitating the introduction of peptides into cells can involve, for example, the attachment of antennapedia or RGD peptide sequences to a NEK2 peptide substrate described herein.

The following are examples of the practice of the invention. They are not to be construed as limiting the scope of the invention in any way.

Examples

Example 1 : Materials and Methods

Anti-phospho serine/threonine monoclonal antibodies were purchased from Upstate Biotechnologies (Lake Placid, NY) and anti-phospho threonine polyclonal antibodies were purchased from Cell Signaling Technologies (Beverly, MA). In addition, a customized anti-phospho c-NAPl antibody was raised to the phosphorylated 11-mer peptide by Sigma-Genosys, Inc (The Woodlands, TX) and double affinity purified. Streptavidin-APC, LANCE™ EU-labeled anti-phospho serine/threonine antibody, and DELFIA® EU-labeled Goat anti-rabbit secondary antibodies were purchased from Perkin Elmer Life Sciences (Emeryville, CA). Active Nek2 enzyme (specific activity = 1065 U/mg; one unit of Nek2 activity is defined as lnmol phosphate incorporated into 0.33 mg/ml myelin basic protein per minute at 30⁰C with a final ATP concentration of 100 μM) was purchased from Upstate Biotechnologies (Lake Placid, NY). Commercially available kinase inhibitors were purchased from Calbiochem (San Diego, CA).

Peptide Design and Synthesis

Arrays included a total of 144 peptides. 11-mer sequences were designed around serine and threonine sites that correspond to phosphorylation sites of proteins that have been reported as physiological substrates for Nek2. Each potential peptide substrate was represented by both a wild-type sequence and a corresponding sequence with an alanine residue substituted at the putative serine/threonine phosphorylation site: peptides #1-19 correspond to Hec-1 amino acid sequences with serine and threonine sites, and with alanine substitutions at these sites (Chen etal. (2002) J. Biol. Chem. 277:49408-16); peptides #20-32 correspond to S6K sequences with serine sites, serine substituted for threonine, and with threonine sites, respectively (Fry et al. (1995) J. Biol. Chem. 270: 12899-905); peptides #33-38 correspond to PPl sequences with threonine sites and with alanine substitutions at these sites (Helps et al. (2000) Biochem. J. 349:509-18); peptides #39-42 correspond to Phospholemma peptide (amino acids 64-72) with serine, threonine, and alanine substitutions, respectively (Fry et al. (1995) J. Biol. Chem. 270:12899-905); peptides #43-111 correspond to C-Nap-1 with serine and threonine sites (Fry et al. (1998) J Cell Biol. 141:1563-74); peptides #112-127 correspond to NIP-I with threonine sites only (Yoo et al. (2004) Exp Cell Res. 292:393-402); and peptides #128- 144 correspond to Nek2 autophosphorylation sites, threonine only (Fry et al. (1999) J. Biol. Chem. 274:16304-10).

Peptide spot synthesis was performed using cellulose membranes derivatized with polyethylene glycol and Fmoc-protected amino acids that were purchased from Intavis Bioanalytical Instruments (San Marcos, CA). Fmoc-protected β-alanine was purchased from Fluka Chemika. The arrays were generated on the membranes by coupling a β-alanine spacer and peptides were synthesized using standard DIC/HOBt coupling chemistry as described (Frank (2002) J Immunol Methods 267:13-26; and Molina (1996) Pept Res. 3:151-55). Activated amino acids were spotted using an Abimed Autospot ASP 222 robot while the washing and Fmoc deprotection steps were done manually. Following the final synthesis cycle, the filters were treated with acetic anhydride (2% in DMF) and side chains were deprotected in 50% trifluoroacetic acid in dichloromethane with 3% triisopropyl silane and 2% water, resulting in an array of peptides that are N- terminally acetylated and attached via the C-terminus. Following side chain deprotection, the filters were washed 2 x 1 minute in ethanol and allowed to dry, then stored in desiccant at —20 degrees C until needed.

Kinase Assays: γ-³³P-ATP Incorporation

Washing in methanol for 10 minutes at room temperature rehydrated the cellulose membranes containing the peptide arrays. The membranes were then equilibrated in enzyme assay buffer for 10 minutes at room temperature (50 mM HEPES, pH=7.5, 10 fflM MgCl₂, 1 mM DTT, 10 uM ATP, 0.5 mM sodium ortho-vanadate, 10 mM β-glycerol phosphate, 0.2 mM EDTA). Membranes were switched into fresh assay buffer with 100 μCi radiolabeled γ-³³P-ATP from ICN Biomedicals, Inc. (Costa Mesa, CA). One membrane was incubated in 0.5 μg/ml Nek2 enzyme (Final [Nek2] = 9.5 nM, final radio specificity = 1.0 Ci/mmol) from UB for 30 minutes at room temperature by gentle shaking. Identical conditions were used for the other membrane, except no enzyme was added (control) (final radiospecificity = 1.0 Ci/mmol). The membranes were washed 3 x 3 minutes with Wash Buffer #1 (2.5 MNaCl, 1% v/v phosphoric acid, 0.5% Triton-X) and 3 x 3 minutes with Wash Buffer #2 (0.5 M NaH₂PO₄, 0.5 M Na₂HPO₄, 1 mM cold ATP). The membranes were then washed 2 x 1 minute with ethanol. Membranes were allowed to dry and then imaged using a Biorad Molecular Imager FX (Hercules, CA).

Kinase Assays: Antibody Detection

Membranes were rehydrated in methanol for 10 minutes at room temperature, and then equilibrated in enzyme assay buffer for 10 minutes at room temperature (50 mM HEPES, pH=7.5, 10 mM MgCl₂, 1 mM DTT, 1 mM ATP, 0.5 mM sodium-ortho- vanadate, 1 mM β-glycerol phosphate, 0.2 mM EDTA). Membranes were switched into fresh assay buffer, and incubated for 30 minutes at room temperature by gentle shaking in either 0.25 μg/ml Nek2 enzyme from UB, or no enzyme (control) (Final [Nek2] = 4.75 nM). Membranes were washed 4 x 2 minutes in TBS, and then blocked for 10 minutes at room temperature in Blocker Casein in TBS #37532 (1% Casein) from Pierce (Rockford, IL). They were then incubated for 1 hr at room temperature with either 1 : 1000 dilution in TBS of phospho-threonine antibody, rabbit polyclonal from Cell Signaling Technology (CST) or 1:1000 dilution in TBS of anti-phospho-serine/threonine mixed mouse monoclonal IgGs from Upstate Biotechnologies (UB). Following the antibody binding, the membranes were washed 3 x 2 minutes with TBS, and then incubated for 20 minutes at room temperature with 1:1000 dilution in Blocker Casein in TBS of either ImmunoPure Goat Anti-Rabbit IgG (H+L), Peroxidase Conjugated from Pierce (Rockford, IL), or ImmunoPure Goat Anti-Mouse IgG (H+L), Peroxidase Conjugated from Pierce (Rockford, IL). Membranes were washed 1 x 5 minutes in Blocker Casein in TBS (1% Casein) from Pierce (Rockford, IL) and 3 x 3 minutes in TBS. Membranes were visualized using SuperSignal West Dura Extended Duration Substrate from Pierce (Rockford, IL) and FluorChem CCD camera from Alpha Innotech (San Leandro, CA).

HPLC Analysis of Peptide Phosphorylation

The Nek2 kinase reaction was carried out in a 96-weIl polypropylene plate (Nunc) by addition of Nek2 [4 ug/ml (75 nM)] to a mixture of peptide (50 μM) and ATP (500 μM) in an assay buffer containing 20 mM HEPES, pH 7.4, 100 mM NaCl, 20 mM MgC12, 2 mM Cys, and 0.005% Brij 35(W/V). The reaction was quenched with 100 mM EDTA (final) at various reaction time points. The mixture (40 μL) was injected onto a Cl 8 reverse phase analytical column (YMC ODS-A, S 5 μm, 120 A, 4.6 x 250 mm) using an Agilent 1100 HPLC instrument. The peptide substrate and phosphorylated product were separated with a 10-60 % acetonitrile/H2O gradient containing 0.1 % TFA over 10 minutes \yith a flow rate of 1 mL/min. Elution was monitored by absorbance at 220 nm and the % of phospho-peptide formation was calculated using the peak area. Three biotinylated peptide substrates, 11-mer, 16-mer, 21-mer (Table 1), were examined using the HPLC analysis to compare the formation of phospho-peptide product.

Evaluation of Inhibition of Nek2 Activity using HPLC Analysis

Compounds were examined for inhibition of Nek2 using HPLC analysis. The reaction was initiated by addition of Nek2 (4 μg/ml) to a mixture of compound, peptide substrate (Biotin-c-NAPl 11-mer, 50 μM) and ATP (500 μM) in a 96-well plate. The reaction was stopped by addition of 100 mM EDTA (final) after 60 minutes. The formation of phospho-11-mer product at varying compound concentrations was monitored with HPLC using the same separation method as described above. The % of phopho-11-mer product was calculated using the peak area and the % of inhibition was determined, accordingly, using equation 1 , % Inhibition = 100 * (1 - Pi / P₀) (eq. 1) where Pi is the % of phopho-11-mer product in the presence of an inhibitor, Po is the % of phopho-11-mer product without an inhibitor.

The % inhibition was plotted as a function of inhibitor concentration, I₀, and the /C₅₀ value was determined by fitting the data to equation 2,

% Inhibition = 100 I₀ ⁿ / (I_o ⁿ + /C₅₀") (eq. 2) where n is the hill constant.

Detection of c-NAPl Phospho-Peptides using LANCE™

Three biotinylated phospho-peptides, p-11-mer, p-16-mer, and p-21-mer (Table 1) were examined using a LANCE™ detection method to evaluate the signal sensitivity with Eu-anti-phospho Ser/Thr UB antibody (ADO 176, Perkin Elmer). Each phospho- peptide, at various concentrations diluted in the assay buffer, was mixed with streptavidin-allophycocyanin (SA-APC, 50 nM final, Perkin Elmer) and Eu-anti-phospho Ser/Thr antibody (1 nM final, ADOl 76, Perkin Elmer) in a black 384-well polypropylene plate (Matrical) to construct a standard curve. The total volume after addition of detection reagents was 20 uL. The time-resolved fluorescence signals at 615 nm and 665 nm wavelengths obtained upon excitation at 340 nm were measured using an Envision plate reader (Perkin Elmer). The signal ratio of 665/615 (APC/Eu) was used in all data analyses.

Determination of Nek2 Activity with LANCE™ Detection

The Nek2 kinase reactions were carried out in a black 384-well polypropylene plate by addition of Nek2 (2 μg/ml) to the mixture of Biotin-peptide substrate (0.5 μM) and ATP (100 μM). The reaction volume was kept to 10 μL. The reaction was stopped with EDTA (5 μL, 40 mM, 1:1 ratio with Mg ²⁺) at various reaction time points. The formation of phospho-peptide product was detected by addition of 5 μL of 4x detection buffer [200 nM streptavidin-APC, 4 nM Eu-anti-phospho Ser/Thr antibody (Perkin Elmer)]. The final concentrations for EDTA, streptavidin-APC, and Eu-anti-phospho Ser/Thr antibody were 10 mM, 50 nM, and 1 nM, respectively. The time-resolved fluorescence signals at 615 nm and 665 nm wavelengths upon excited at 340 nm was measured using an Envision plate reader (Perkin Elmer). The signal ratio of 665/615 (APC/Eu) was used in all data analyses. Various assay conditions, such as the concentrations of peptide substrate, Nek2, EDTA, SA-APC, Eu-antibody, and DMSO₃ were examined to optimize the Nek2 LANCE™ assay.

Evaluation of Inhibition of Nek2 Activity using LANCE™ Assay Format

The same compounds tested in the HPLC assay were evaluated in the Nek2 LANCE™ assay to determine the IC5₀ values for each compound. Kinase reaction and detection were carried out similarly as described above. Nek2 (2 μg/ml) was added to the mixture of an inhibitor, Biotin-peptide substrate (16-mer, 0.5 μM), and ATP (100 μM) and further incubated for 2 hours at room temperature. The % inhibition was calculated using equation 3,

% Inhibition = 100 * ( 1 - (S; - S_b) / (S₀ - S_b)), (eq. 3) where Si is the signal ratio of 665/615 in the presence of inhibitor, So is the signal ratio of 665/615 in the absence of inhibitor (enzyme control), and S_b is the background signal ratio.

The % inhibition was plotted as a function of inhibitor concentration and the /C50 value was determined by fitting the data to equation 2.

DELFIA® Assay: c-NAPl Peptide Phosphorylation with Custom Antibodies

The Nek2 activity assay was carried out by addition of Nek2 (2 μg/ml) to the mixture of Biotin-peptide substrate (0.5 μM) and ATP (100 μM) in an assay buffer (20 mM HEPES, 7.4, 100 mM NaCl, 20 mM MgC12, 2 mM cysteine and 0.05% Brij 35). The reaction was stopped with EDTA (13.3 mM, 1: 1 ratio with Mg ²⁺) after a 2-hour incubation, and 10 μL of the reaction mixture was applied to a Streptavidin coated 96- well plate from Perkin Elmer. After a 1-hour incubation the plate was washed with H₂O and then with a blocking buffer (50 mM Tris.Cl, pH 7.4, 100 mM NaCl, 0.01% Tween 20, 1% BSA) to block non-specific protein binding surfaces. Two different primary anti- phospho Ser/Thr antibodies in a blocking buffer (100 ng/ml), one from Upstate (mixed mouse monoclonal IgGs) and the other from Sigma (custom-made, rabbit polyclonal), were used for detection of phospho peptide. After a 1-hour incubation the secondary Eu- labeled DELFIA® antibodies (anti-mouse or anti-rabbit, 100 ng/ml, Perkin Elmer) were added to the plates and incubated for an additional 1-hour. The plates were then washed with buffer (50 mM Tris.Cl, pH 7.4, 100 mM NaCl, 0.01% Tween 20). The DELFΪA® enhancement solution (100 μL) was added and the time-resolved fluorescence signal was measured at 615 ran excited at 340 nm using an Envision plate reader. The Nek2 inhibition assays were carried out similarly by addition of Nek2 (2 μg/ml) to the mixture of Biotin-peptide substrate (0.5 μM), ATP (100 μM), and inhibitors.

Detection of Nek2 Autophosphorylation by DELFIA®

Increasing concentrations (0.02, 0.1, 0.2, 0.5, 1.0 and 2.0 ug/ml) of Nek2 (Upstate Biotechnology, Lake Placid, NY) were incubated with 100 uM ATP (Rm) in assay buffer (20 mM HEPES, 7.4, 100 mM NaCl, 20 mM MgC12, 2 mM cysteine and 0.05% Brij 35) for 1 hour in streptavidin-coated polystyrene 96-well plates (Perkin Elmer). The reactions were terminated with 20 mM EDTA for 10 minutes and solutions discarded. Plates were washed 3x with Ix TBS and blocked for 1 hour in 5% BSA in TBS/0.1% Tween 20 (TBST). Phosphorylated Nek2 was detected with a 1 : 1000 dilution of polyclonal anti-phospho-Thr CST primary antibody for 1 hour at room temperature, followed by a 1 :5000 dilution of DELFIA® Europium-labeled secondary antibody for an additional 1 hour at room temperature. Fluorescence energy transmission was measured at 615 nm on a Victor™ time-resolved fluorometer (Perkin Elmer).

Detection of Nek2 Autophosphorylation by Western Blotting

A total of 20 ug/ml of Nek2 (Upstate Biotechnology) was incubated in the presence or absence of 100 μM ATP for 1 hour in assay buffer and separated on a 10% Bis-Tris SDS gel under non-denaturing conditions. Enzyme was transferred onto nitrocellulose and phosphorylated enzyme was detected with the CST polyclonal anti- pThr antibody. Membranes were re-probed with anti-Nek2 monoclonal antibodies (BD Biosciences) to ensure equal protein loading

Example 2: Identification of Nek2 Peptide Substrates

Two antibodies were used to develop a rapid and sensitive non-radioactive assay to detect phosphorylation of peptide substrates by Nek2: a mouse monoclonal antibody from Upstate Biotechnology ("UB") and a rabbit polyclonal antibody from Cell Signaling Technology ("CST") that detect phospho-serine and threonine residues. An array of 144 11-mer sequences were designed around serine and threonine sites, corresponding to potential phosphorylation sites of reported physiological substrates for Nek2 (Chen et al. (2002) J. Biol. Chem. 277:49408-16; Helps et al. (2000) Biochem J. 349:509-18; Yoo et al. (2004) Exp Cell Res. 292:393-402; Fry et al. (1999) J. Biol. Chem. 274:16304-10).

Peptide arrays were synthesized on nitrocellulose membranes and probed with the CST anti-phospho-threonine antibody. Of the peptides scanned with the CST antibody, several peptides containing threonine residues located within the region of c-NAPl bounded by amino acids 1851 to 2442 or the region of Nek2 bounded by amino acids 1 to 222 consistently generated positive signals when compared to the no enzyme control (Table 1). In particular, Nek2 phosphorylated nine c-NAPl 11 -mer peptides, with peptide #97 (amino acids 2213-2223; DELELTRRALE; SEQ ID NO:6) generating the strongest signal. In addition, out of the six NEK2 peptides that generated positive signals, particularly strong signals were detected with peptides #139 (amino acids 165- 175), #140 (amino acids 170-180), and #142 (amino acids 212-222) (Table 1).

Table 1: c-NAPl and Nek2 Phosphorylation Sites Detected by CST Anti-Phospho-Threonine Antibodies

When the arrays were scanned with the UB antibody, peptide #97 of c-NAPl generated a positive signal compared to the no-enzyme control, suggesting that the phosphorylated product may be a preferred antigen for the UB anti-phospho- serine/threonine antibody. The latter was confirmed by incorporation of the threonine- only containing peptide DELELTRRALE (SEQ ID NO:6) and not the alanine substituted control peptide DELELARRALE (SEQ ID NO: 18) with γ-³³P phosphate. Reprobe of the γ-³³P blot with the UB antibody confirmed recognition of phospho-c-NAP 1 peptides.

Example 3: Detection of c-NAPl Phosphorylation by HPLC

To confirm that peptide #97 (SEQ ID NO:6) was a substrate for Nek2 in solution, an in vitro kinase assay was performed and the phosphorylated product was analyzed by HPLC. Nearly 30% of peptide #97 was converted into product within 1.5 hours of incubation with Nek2 enzyme. In addition, variants of peptide #97, prepared by Anaspec, Inc. (San Jose, CA), were tested by increasing the amino acids by 5 (16-mer; SEQ ID NO: 16) and 10 (21-mer; SEQ ID NO: 17) at the amino-terminus in order to optimize the substrate for the TR-FRET assay (Table 2). In Table 2, the underlined and bolded threonine residue represents the only phosphorylation site within the substrates. While Nek2 phosphorylated both the 16-mer and 21-mer peptides, the 16-mer generated greater than 50 % product within 1.5 hours of incubation with Nek2. Thus, peptide #97 and peptides with at least 10 amino acids added to the N-terminus of peptide #97 function as substrates of Nek2.

Table 2: Biotinylated c-NAPl Peptide Substrates Detected by UB Anti-Phospho

Serine/Threonine Antibodies

Example 4: Detection of c-NAPl Phosphorylation by LANCE™ Assay

The LANCE™ assay is useful for high throughput screening (HTS) of small- molecule inhibitors because, e.g., it requires minimal handling steps (i.e., is homogenous), is non-radioactive, and has a high Z'-factor. The inability of the UB antibody to detect the tested Nek2 peptides suggested that this antibody would be useful for a homogenous assay such as the LANCE™ assay in which the Nek2 enzyme would be present during the detection phase.

The three biotinylated variants of peptide #97 depicted in Table 2 were tested using an Eu-labeled UB antibody as the energy donor for resonance transfer. The 16-mer and 21-mer provided the greatest signal to noise ratio with increasing concentrations of peptide, while the 11-mer did not give any signal in the LANCE™ assay. The 16-mer was the optimal substrate in the LANCE™ assay, achieving a 7 fold increase above the background signal to noise ratio at ideal reagent concentration. The optimal enzyme reaction time was 2 hours for the 16-mer peptide and was within the linear range with a Kni_app for ATP of 111 μM. The addition of EDTA and signal stability as a function of time was also tested using the 16-mer peptide to ensure that detection times greater than 30 minutes were feasible given that this may be the amount of time that may elapse during sample analysis. The recommended concentration of EDTA (10 mM 1:1 with Mg2+) effectively stopped the enzyme reaction and the LANCE™ signal was stable during the incubation. Increasing the concentration of EDTA to 15 and 20 mM reduced the signal/noise ratio and the FRET signal of the APC acceptor from 8 fold to about 4 fold.

Example 5: Detection of Phosphorylated c-NAPl Peptides bv DELFIA®

Although the LANCE™ assay is useful and efficient for HTS, the homogeneous nature of the assay can yield false positive due to fluorescent compounds. A secondary assay was thus developed to confirm hits obtained from the LANCE™ assay. The DELFIA® assay is a non-homogenous ELISA-based assay that, due to its multiple washing steps, removes unbound compounds after incubation with enzyme. The original 11-mer peptide #97 was tested in a DELFIA® based assay using a customized polyclonal antibody (Sigma-Genosys) raised to the phosphor-threonine residue of peptide #97 and yielded a 27-fold increase above the no enzyme control. More importantly, the signal/background ratio of the Sigma-Genosys antibody was 10-fold greater than the UB antibody. This DELFIA® assay thus constitutes a robust secondary assay to evaluate inhibitors of Nek2 using a physiologically relevant substrate.

Example 6: Detection of Nek2 Autophosphorylation by DELFIA®

An alternative DELFIA® secondary assay was also developed to detect Nek2 autophosphorylation that occurs upon increased expression during S-phase of the cell cycle. Because the CST antibody recognized several peptide residues in the Nek2 protein (Table 1), this antibody was used in an attempt to detect phosphorylated Nek2 in an in vitro kinase assay. Increasing concentrations of Nek2 up to 2 ug/ml resulted in a 60 fold increase above the ATP minus control. To confirm that the CST antibody was detecting phosphorylated Nek2, the in vitro kinase reactions were run in the presence or absence of ATP and, after terminating the reactions with 10 mM EDTA, products were analyzed by Western blotting. A single band was detected at 42 kd (the molecular weight of Nek2) with the CST antibody only in the ATP plus reactions and not in the ATP minus control. Re-probing the blots with anti-Nek2 mono-specific antibodies confirmed that Nek2 was indeed present in equal amounts. Thus, the CST antibody can be used in a DELFIA® based assay to screen for Nek2 autophosphorylation.

Example 7: Identification of Potent Nek2 Inhibitors by HPLC, LANCE™ and DELFIA®

The Nek2 autophosphorylation DELFIA® assay was validated by screening a library of approximately 12,000 compounds. Three potent inhibitors (ICs₀ 50 to 231 nM) were identified. The potency of the compounds was confirmed by screening in the four assays described herein: HPLC₅ LANCE™ and DELFIA® (with c-NAPl peptide and with Nek2 only). Minimal variation in IC₅₀ values was observed regardless of the assay used, thereby confirming not only that the compounds were indeed Nek2 inhibitors but that the assays were compatible. Thus, all four assays are capable of identifying and validating potent Nek2 inhibitors and can be effectively used to screen libraries. Other Embodiments

While the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

What is claimed is:

1. An isolated peptide that is less than 50 amino acids in length and comprises SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 11 , SEQ ID NO: 12, SEQ ID NO:13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, or a variant thereof, wherein the variant is a phosphorylation substrate of NEK2.

2. The peptide of claim 1, wherein the peptide comprises SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ IDNO:14, SEQ ID NO.15, SEQ ID NO:16, or SEQ ID NO:17.

3. The peptide of claim 1, wherein the peptide comprises a variant of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 11 , SEQ ID

NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, or SEQ ID NO: 17 in which at least one but not more than five amino acid residues are substituted, deleted, or inserted.

4. The peptide of claim 1, wherein the peptide comprises a variant of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ED NO:8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 11 , SEQ ID NO: 12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, or SEQ ID NO: 17 in which at least one but not more than two amino acid residues are substituted, deleted, or inserted.

5. The peptide of any of claims 1 to 4, wherein the peptide is less than 40 amino acids in length.

6. The peptide of any of claims 1 to 4, wherein the peptide is less than 30 amino acids in length.

7. The peptide of any of claims 1 to 4, wherein the peptide is less than 25 amino acids in length.

8. The peptide of any of claims 1 to 4, wherein the peptide is 20 or fewer amino acids in length.

9. The peptide of any of claims 1 to 4, wherein the peptide is 15 or fewer amino acids in length.

10. The peptide of any of claims 1 to 9, wherein the peptide comprises a heterologous amino acid sequence.

11. The peptide of claim 1, wherein the peptide consists of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ED NO.4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, or SEQ ID NO:17.

12. The peptide of any of claims 1 to 11, wherein the peptide is phosphorylated on a serine or threonine residue.

13. An isolated antibody that specifically binds to a peptide whose amino acid sequence consists of SEQ ED NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NOrIO, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID N0:15, SEQ ID NO: 16, or SEQ ID NO: 17.

14. The antibody of claim 13, wherein the antibody preferentially binds the peptide when phosphorylated on a serine or threonine amino acid residue.

15. The peptide of any of claims 1 to 11, wherein the peptide is conjugated to a first member of a specific binding pair.

16. The peptide of claim 15, wherein the first member of a specific binding pair is biotin.

17. The peptide of any of claims 1 to 11, wherein the peptide is conjugated to a detection moiety.

18. The peptide of claim 17, wherein the detection moiety is a fluorescent agent.

19. A method of generating an immune response in a mammal, the method comprising administering to the mammal an effective amount of the peptide of any of claims 1-12.

20. A method of detecting kinase activity of a NEK2 protein, the method comprising: contacting a NEK2 protein with the peptide of any of claims 1 to 11 or 15-18 under conditions effective to permit phosphorylation of the peptide; and measuring phosphorylation of the peptide, wherein phosphorylation of the peptide indicates kinase activity of theNEK2 protein.

21. A method of identifying a compound that inhibits phosphorylation of a NEK2 substrate, the method comprising: contacting, in the presence of a candidate compound, a NEK2 protein with the peptide of any of claims 1 to 11 or 15-18; and measuring phosphorylation of the peptide, wherein decreased phosphorylation of the peptide in the presence of the candidate compound as compared to phosphorylation of the peptide that occurs in the absence of the candidate compound indicates that the candidate compound inhibits phosphorylation of a NEK2 substrate by the NEK2 protein.

22. The method of claim 20 or 21, wherein measuring phosphorylation of the peptide comprises: contacting the peptide with an antibody that (i) is conjugated to a first fluorescent agent and (ii) specifically binds to the peptide when the peptide is phosphorylated on a serine or threonine residue, wherein the peptide is conjugated to a second fluorescent agent; and detecting the occurrence of fluorescence resonance energy transfer between the first fluorescent agent and the second fluorescent agent as an indicator of phosphorylation of the peptide.

23. The method of claim 20 or 21, wherein measuring phosphorylation of the peptide comprises: contacting the peptide with an antibody that (i) is conjugated to a detection moiety and (ii) specifically binds to the peptide when the peptide is phosphorylated on a serine or threonine residue; removing antibody that is not bound to the peptide; and detecting the detection moiety associated with the peptide as an indicator of phosphorylation of the peptide.

24. The method of claim 20 or 21, wherein measuring phosphorylation of the peptide comprises passaging the peptide through a stationary phase, wherein retardation of the peptide during passage through the stationary phase indicates the phosphorylation status of the peptide.

25. A method of inhibiting phosphorylation of a NEK2 substrate, the method comprising contacting a NEK2 protein with a composition that that binds to the NEK2 protein and inhibits phosphorylation of a NEK2 substrate by the NEK2 protein.

26. The method of claim 25, wherein the composition is a peptide.

27. The method of claim 26, wherein the peptide is less than 50 amino acids in length and comprises SEQ ID NO: I₃ SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11₅ SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO: 16, SEQ ID NO: 17, or a variant thereof, wherein the variant is a phosphorylation substrate of NEK2.