Patent Application
20090004180
The Rho family of small GTPases comprises a group of about 20 mammalian proteins, which include Rho, Rac, and CDC42. These ubiquitous and highly conserved proteins are key regulators of many cellular processes, including cell migration and shape changes, cell-cell and cell-matrix adhesion, cell-cycle progression and cytokinesis, and gene expression (see Burridge K and Wennerberg (2004) Cell 116(2):167-179). Like their close relative Ras, Rho GTPases act as molecular switches that cycle between a biochemically-active GTP-bound state and an inactive GDP-bound state. Regulation of cycling is mediated predominantly through 3 classes of proteins: guanine exchange factors (GEFs) which promote the active GTP-bound state by facilitating exchange of GDP for GTP; GTPase-activating proteins (GAPs) which promote the inactive state by stimulating the relatively weak intrinsic hydrolase activity of GTPases; and guanine-dissociation inhibitors (GDIs) which bind to and sequester GTPase in their GDP-bound form. Activation of many GEFs and their target GTPase occurs in response to a variety of upstream stimuli such as growth factor engagement and cell-cell or cell-matrix adhesion. As regulators, activated Rho proteins bind and usually activate one or more of a variety of downstream effecter proteins (e.g., rho kinases, formins, p21-activated kinases) that elicit a cellular response.
Potential roles of Rho proteins in promoting cancer progression have been suggested based on the well-established functions of individual family members in promoting cell-cycle progression and cellular motility and tissue invasion, as well of the oncogenic properties associated with different GEFs acting on Rho proteins. Constitutively-activated forms of many GEFs result in hyperactivation of one or more Rho family members and are oncogenic for rodent cells. Further, overexpression of specific Rho proteins or Rho effectors has been observed for several human cancers, including breast, lung, and colorectal (see Croft D R et al (2004) Can Res 64:8994-9001, and references therein). In several cases, in vivo animal model studies have provided compelling evidence that overexpression of either a Rho protein or effector contributes significantly to tumor cell invasiveness and/or metastasis (See Sahai, E. (2005) Cur Opin Genet & Dev. (2005) 15:87-96).
Studies of model systems such as C. elegans and Drosophila have identified invertebrate orthologues of Rho, Rao, and CDC42 that show conservation in function as well as structure. C. elegans contains 3 Rac-encoding genes (ced-10, mig-2, rac-2) and single genes encoding CDC42 (cdc-42) or Rho (rho-1). The three nematode Rac genes have redundant roles in cell motility and migration (Lundquist E A et al., Development (2001) 128(22):4475-88), while rho-1 is required for cytokinesis (Jantsch-Plunger V et al (2000) J Cell Biol 149(7):1291-404) and cdc-42 has an essential role in cell polarity (Gotta M et al (2001) Curr Biol 11(7):482-488). C. elegans orthologues of several mammalian GEFs have also been characterized, including Dock180/ELMO (ced-2/ced-12) and Ect2 (let-21). These particular GEFs appear conserved in function as well as they exhibit loss-of-function mutant phenotypes that overlap those associated with Rho, Rac, or CDC42 mutants. For example, like their mammalian orthologues, C. elegans Rho (rho-1) and Ect2 (let-21) are required at the end of mitosis for cleavage furrow ingression. Reflecting this role, cytokinesis-defective phenotypes are observed in reduction-of-function let-21 mutants or after RNA inhibition (RNAi) of either let-2 or rho-1 (R. Francis, unpublished observations; T. Schedl, personal communication).
KIF23 (kinesin family member 23) is a member of kinesin-like protein family. This family includes microtubule-dependent molecular motors that transport organelles within cells and move chromosomes during cell division. KIF23 has been shown to cross-bridge antiparallel microtubules and drive microtubule movement in vitro. Alternate splicing of this gene results in two transcript variants encoding two different isoforms.
PPIE (peptidylprolyl isomerase E; cyclophilin E) is a member of the peptidyl-prolyl cis-trans isomerase (PPIase) family. PPIases catalyze the cis-trans isomerization of proline imidic peptide bonds in oligopeptides and accelerate the folding of proteins. PPIE contains a highly conserved cyclophilin (CYP) domain as well as an RNA-binding domain, and possesses PPIase and protein folding activities and also exhibit RNA-binding activity.
FDPS (farensyl diphosphate synthase) catalyzes the conversion of geranyl diphosphate and isopentenyl diphosphate to diphosphate and trans, trans-farnesyl diphosphate in the isoprene biosynthetic pathway.
Phosphatidylinositol (PI) 4-kinase (PIK4CA) catalyzes the first committed step in the biosynthesis of phosphatidylinositol 4,5-bisphosphate. The mammalian PI 4-kinases have been classified into two types, II and III, based on their molecular mass, and modulation by detergent and adenosine. Two transcript variants encoding different isoforms have been described for this gene.
Protein kinases with no lysine (lysine deficient protein kinase; PRKWNK) are cytoplasmic serine-threonine kinases that contain cysteine in place of lysine at a conserved location, yet have kinase activity. Mutations in two of PRKWNKs, the WNK1 and WNK4, cause type II pseudohypoaldosteronism, an autosomal dominant disorder featuring hypertension, hyperkalemia, and renal tubular acidosis.
Intercellular communication is often mediated by receptors on the surface of one cell that recognize and are activated by specific protein ligands released by other cells. Members of one class of cell surface receptors, receptor tyrosine kinases (RTKs), are characterized by having a cytoplasmic domain containing intrinsic tyrosine kinase activity. This kinase activity is regulated by the binding of a cognate ligand to the extracellular portion of the receptor. RTKs are expressed in cell type-specific fashions and play a role critical for the growth and differentiation of those cell types. ROR1 (Neurotrophic tyrosine kinase receptor related 1) is expressed in neural tissues and may be involved in transmembrane receptor protein tyrosine kinase signaling pathways (Oishi, I., et al (1999) Genes Cells 4:41-56; Masiakowski, P., and Carroll, R. D. (1992) J Biol Chem 267:26181-90; Reddy, U. R., et al (1996) Oncogene 13:1555-9). ROR2 (Receptor tyrosine kinase-like orphan receptor 2) is another neuronal-specific member of the RTK family. Mutations in ROR2 are associated with skeletal disorders, including dominant brachydactyly type BI and recessive Robinow syndrome (Afzal, A. R., et al (2000) Nat Genet 25:419-22; Oldridge, M., et al (2000) Nat Genet 24:275-8).
MELK (maternal embryonic leucine zipper kinase) is a member of the evolutionarily conserved KIN1/PAR-1/MARK kinase family which is involved in cell polarity and microtubule dynamics.
The ability to manipulate the genomes of model organisms such as C. elegans provides a powerful means to analyze biochemical processes that, due to significant evolutionary conservation, have direct relevance to more complex vertebrate organisms. Due to a high level of gene and pathway conservation, the strong similarity of cellular processes, and the functional conservation of genes between these model organisms and mammals, identification of the involvement of novel genes in particular pathways and their functions in such model organisms can directly contribute to the understanding of the correlative pathways and methods of modulating them in mammals (see, for example, Dulubova I, et al, J Neurochem 2001 April; 77(1):229-38; Cai T, et al., Diabetologia 2001 January; 44(1):81-8; Pasquinelli A E, et al., Nature. 2000 Nov. 2; 408(6808):37-8; Ivanov I P, et al., EMBO J 2000 Apr. 17; 19(8); 1907-17; Vajo Z et al., Mamm Genome 1999 October; 10(10):1000-4). For example, a genetic screen can be carried out in an invertebrate model organism having underexpression (e.g. knockout) or overexpression of a gene (referred to as a “genetic entry point”) that yields a visible phenotype. Additional genes are mutated in a random or targeted manner. When a gene mutation changes the original phenotype caused by the mutation in the genetic entry point, the gene is identified as a “modifier” involved in the same or overlapping pathway as the genetic entry point. When the genetic entry point is an ortholog of a human gene implicated in a disease pathway, such as RHO, modifier genes can be identified that may be attractive candidate targets for novel therapeutics.
All references cited herein, including patents, patent applications, publications, and sequence information in referenced Genbank identifier numbers, are incorporated herein in their entireties.
We have discovered genes that modify the RHO pathway in C. elegans, and identified their human orthologs, hereinafter referred to as Modifiers of RHO (RHO). Specifically, we have discovered that one gene, KIF23 (kinesin family member 23) modifies the RHO pathway in a number of human tissues and cell lines. This gene is also known in the literature as MKLP1 (mitotic kinesin-like protein 1). The encoded protein contains an amino terminal kinesin-motor domain, followed by a neck region, a stalk region important for dimerization and NLS domains near the carboxy-terminus. The encoded protein bridges anti-parallel microtubules (MT) that form part of the central spindle during cytokinesis. The encoded protein directs the recruitment of factors such as Ect2 and RhoA that drive cleavage furrow formation and ingression. The encoded protein also functions in other cell structures that express anti-parallel MTs as well. The invention provides methods for utilizing these RHO modifier genes and polypeptides to identify KIF23-modulating agents that are candidate therapeutic agents that can be used in the treatment of disorders associated with defective or impaired RHO function and/or KIF23 function. Preferred KIF23-modulating agents specifically bind to KIF23 polypeptides and restore RHO function. Other preferred KIF23-modulating agents are nucleic acid modulators such as antisense oligomers and RNAi that repress KIF23 gene expression or product activity by, for example, binding to and inhibiting the respective nucleic acid (i.e. DNA or mRNA).
KIF23 modulating agents may be evaluated by any convenient in vitro or in vivo assay for molecular interaction with a KIF23 polypeptide or nucleic acid. In one embodiment, candidate KIF23 modulating agents are tested with an assay system comprising a KIF23 polypeptide or nucleic acid. Agents that produce a change in the activity of the assay system relative to controls are identified as candidate RHO modulating agents. The assay system may be cell-based or cell-free. KIF23-modulating agents include KIF23 related proteins (e.g. dominant negative mutants, and biotherapeutics); KIF23-specific antibodies; KIF23-specific antisense oligomers and other nucleic acid modulators; and chemical agents that specifically bind to or interact with KIF23 or compete with KIF23 binding partner (e.g. by binding to a KIF23 binding partner). In one specific embodiment, a small molecule modulator is identified using a binding assay. In specific embodiments, the screening assay system is selected from an apoptosis assay, a cell proliferation assay, and an angiogenesis assay.
In another embodiment, candidate RHO pathway modulating agents are further tested using a second assay system that detects changes in the RHO pathway, such as angiogenic, apoptotic, or cell proliferation changes produced by the originally identified candidate agent or an agent derived from the original agent. The second assay system may use cultured cells or non-human animals. In specific embodiments, the secondary assay system uses non-human animals, including animals predetermined to have a disease or disorder implicating the RHO pathway, such as an angiogenic, apoptotic, or cell proliferation disorder (e.g. cancer).
The invention further provides methods for modulating KIF23 function and/or the RHO pathway in a mammalian cell by contacting the mammalian cell with an agent that specifically binds a KIF23 polypeptide or nucleic acid. The agent may be a small molecule modulator, a nucleic acid modulator, or an antibody and may be administered to a mammalian animal predetermined to have a pathology associated with the RHO pathway.
A C. elegans genetic screen was designed which employed RNAi of specific genes to identify genetic modifiers of Rho pathway function. Methods for using RNAi to silence genes in C. elegans are known in the art (Fire A, et al., 1998 Nature 391:806-811; Fire, A. Trends Genet. 15, 358-363 (1999); WO9932619). Genes causing altered phenotypes in the worms were identified as modifiers of the RHO pathway, followed by identification of their orthologs. Accordingly, vertebrate orthologs of these modifiers, and preferably the human orthologs, KIF23 genes (i.e., nucleic acids and polypeptides) are attractive drug targets for the treatment of pathologies associated with a defective RHO signaling pathway, such as cancer. Table 1 (Example II) lists the modifiers and their orthologs.
In vitro and in vivo methods of assessing KIF23 function are provided herein. Modulation of the KIF23 or their respective binding partners is useful for understanding the association of the RHO pathway and its members in normal and disease conditions and for developing diagnostics and therapeutic modalities for RHO related pathologies. KIF23-modulating agents that act by inhibiting or enhancing KIF23 expression, directly or indirectly, for example, by affecting a KIF23 function such as enzymatic (e.g., catalytic) or binding activity, can be identified using methods provided herein. KIF23 modulating agents are useful in diagnosis, therapy and pharmaceutical development.
Sequences related to KIF23 nucleic acids and polypeptides that can be used in the invention are disclosed in Genbank (referenced by Genbank identifier (GI) or RefSeq number), shown in Table 1 and in the appended sequence listing.
The term “KIF23 polypeptide” refers to a full-length KIF23 protein or a functionally active fragment or derivative thereof. A “functionally active” KIF23 fragment or derivative exhibits one or more functional activities associated with a full-length, wild-type KIF23 protein, such as antigenic or immunogenic activity, enzymatic activity, ability to bind natural cellular substrates, etc. The functional activity of KIF23 proteins, derivatives and fragments can be assayed by various methods known to one skilled in the art (Current Protocols in Protein Science (1998) Coligan et al., eds., John Wiley & Sons, Inc., Somerset, N.J.) and as further discussed below. In one embodiment, a functionally active KIF23 polypeptide is a KIF23 derivative capable of rescuing defective endogenous KIF23 activity, such as in cell based or animal assays; the rescuing derivative may be from the same or a different species. For purposes herein, functionally active fragments also include those fragments that comprise one or more structural domains of KIF23, such as a kinase domain or a binding domain. Protein domains can be identified using the PFAM program (Bateman A., et al., Nucleic Acids Res, 1999, 27:260-2). Methods for obtaining KIF23 polypeptides are also further described below. In some embodiments, preferred fragments are functionally active, domain-containing fragments comprising at least 25 contiguous amino acids, preferably at least 50, more preferably 75, and most preferably at least 100 contiguous amino acids of a KIF23. In further preferred embodiments, the fragment comprises the entire functionally active domain.
The term “KIF23 nucleic acid” refers to a DNA or RNA molecule that encodes a KIF23 polypeptide. Preferably, the KIF23 polypeptide or nucleic acid or fragment thereof is from a human, but can also be an ortholog, or derivative thereof with at least 70% sequence identity, preferably at least 80%, more preferably 85%, still more preferably 90%, and most preferably at least 95% sequence identity with human KIF23. Methods of identifying orthlogs are known in the art. Normally, orthologs in different species retain the same function, due to presence of one or more protein motifs and/or 3-dimensional structures. Orthologs are generally identified by sequence homology analysis, such as BLAST analysis, usually using protein bait sequences. Sequences are assigned as a potential ortholog if the best hit sequence from the forward BLAST result retrieves the original query sequence in the reverse BLAST (Huynen M A and Bork P, Proc Natl Acad Sci (1998) 95:5849-5856; Huynen M A et al., Genome Research (2000) 10:1204-1210). Programs for multiple sequence alignment, such as CLUSTAL (Thompson J D et al, 1994, Nucleic Acids Res 22:4673-4680) may be used to highlight conserved regions and/or residues of orthologous proteins and to generate phylogenetic trees. In a phylogenetic tree representing multiple homologous sequences from diverse species (e.g., retrieved through BLAST analysis), orthologous sequences from two species generally appear closest on the tree with respect to all other sequences from these two species. Structural threading or other analysis of protein folding (e.g., using software by ProCeryon, Biosciences, Salzburg, Austria) may also identify potential orthologs. In evolution, when a gene duplication event follows speciation, a single gene in one species, such as C. elegans, may correspond to multiple genes (paralogs) in another, such as human. As used herein, the term “orthologs” encompasses paralogs. As used herein, “percent (%) sequence identity” with respect to a subject sequence, or a specified portion of a subject sequence, is defined as the percentage of nucleotides or amino acids in the candidate derivative sequence identical with the nucleotides or amino acids in the subject sequence (or specified portion thereof), after aligning the sequences and introducing gaps, if necessary to achieve the maximum percent sequence identity, as generated by the program WU-BLAST-2.0a19 (Altschul et al., J. Mol. Biol. (1997) 215:403-410) with all the search parameters set to default values. The HSP S and HSP S2 parameters are dynamic values and are established by the program itself depending upon the composition of the particular sequence and composition of the particular database against which the sequence of interest is being searched. A % identity value is determined by the number of matching identical nucleotides or amino acids divided by the sequence length for which the percent identity is being reported. “Percent (%) amino acid sequence similarity” is determined by doing the same calculation as for determining % amino acid sequence identity, but including conservative amino acid substitutions in addition to identical amino acids in the computation.
A conservative amino acid substitution is one in which an amino acid is substituted for another amino acid having similar properties such that the folding or activity of the protein is not significantly affected. Aromatic amino acids that can be substituted for each other are phenylalanine, tryptophan, and tyrosine; interchangeable hydrophobic amino acids are leucine, isoleucine, methionine, and valine; interchangeable polar amino acids are glutamine and asparagine; interchangeable basic amino acids are arginine, lysine and histidine; interchangeable acidic amino acids are aspartic acid and glutamic acid; and interchangeable small amino acids are alanine, serine, threonine, cysteine and glycine.
Alternatively, an alignment for nucleic acid sequences is provided by the local homology algorithm of Smith and Waterman (Smith and Waterman, 1981, Advances in Applied Mathematics 2:482-489; database: European Bioinformatics Institute; Smith and Waterman, 1981, J. of Mol. Biol., 147:195-197; Nicholas et al., 1998, “A Tutorial on Searching Sequence Databases and Sequence Scoring Methods” (www.psc.edu) and references cited therein; W. R. Pearson, 1991, Genomics 11:635-650). This algorithm can be applied to amino acid sequences by using the scoring matrix developed by Dayhoff (Dayhoff: Atlas of Protein Sequences and Structure, M. O. Dayhoff ed., 5 suppl. 3:353-358, National Biomedical Research Foundation, Washington, D.C., USA), and normalized by Gribskov (Gribskov 1986 Nucl. Acids Res. 14(6):6745-6763). The Smith-Waterman algorithm may be employed where default parameters are used for scoring (for example, gap open penalty of 12, gap extension penalty of two). From the data generated, the “Match” value reflects “sequence identity.”
Derivative nucleic acid molecules of the subject nucleic acid molecules include sequences that hybridize to the nucleic acid sequence of KIF23. The stringency of hybridization can be controlled by temperature, ionic strength, pH, and the presence of denaturing agents such as formamide during hybridization and washing. Conditions routinely used are set out in readily available procedure texts (e.g., Current Protocol in Molecular Biology, Vol. 1, Chap. 2.10, John Wiley & Sons, Publishers (1994); Sambrook et al., Molecular Cloning, Cold Spring Harbor (1989)). In some embodiments, a nucleic acid molecule of the invention is capable of hybridizing to a nucleic acid molecule containing the nucleotide sequence of a KIF23 under high stringency hybridization conditions that are: prehybridization of filters containing nucleic acid for 8 hours to overnight at 65° C. in a solution comprising 6× single strength citrate (SSC) (1×SSC is 0.15 M NaCl, 0.015 M Na citrate; pH 7.0), 5×Denhardt's solution, 0.05% sodium pyrophosphate and 100 μg/ml herring sperm DNA; hybridization for 18-20 hours at 65° C. in a solution containing 6×SSC, 1×Denhardt's solution, 100 μg/ml yeast tRNA and 0.05% sodium pyrophosphate; and washing of filters at 65° C. for 1 h in a solution containing 0.1×SSC and 0.1% SDS (sodium dodecyl sulfate).
In other embodiments, moderately stringent hybridization conditions are used that are: pretreatment of filters containing nucleic acid for 6 h at 40° C. in a solution containing 35% formamide, 5×SSC, 50 mM Tris-HCl (pH7.5), 5 mM EDTA, 0.1% PVP, 0.1% Ficoll, 1% BSA, and 500 μg/ml denatured salmon sperm DNA; hybridization for 18-20 h at 40° C. in a solution containing 35% formamide, 5×SSC, 50 mM Tris-HCl (pH7.5), 5 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100 μg/ml salmon sperm DNA, and 10% (wt/vol) dextran sulfate; followed by washing twice for 1 hour at 55° C. in a solution containing 2×SSC and 0.1% SDS.
Alternatively, low stringency conditions can be used that are: incubation for 8 hours to overnight at 37° C. in a solution comprising 20% formamide, 5×SSC, 50 mM sodium phosphate (pH 7.6), 5×Denhardt's solution, 10% dextran sulfate, and 20 μg/ml denatured sheared salmon sperm DNA; hybridization in the same buffer for 18 to 20 hours; and washing of filters in 1×SSC at about 37° C. for 1 hour.
KIF23 nucleic acids and polypeptides are useful for identifying and testing agents that modulate KIF23 function and for other applications related to the involvement of KIF23 in the RHO pathway. KIF23 nucleic acids and derivatives and orthologs thereof may be obtained using any available method. For instance, techniques for isolating cDNA or genomic DNA sequences of interest by screening DNA libraries or by using polymerase chain reaction (PCR) are well known in the art. In general, the particular use for the protein will dictate the particulars of expression, production, and purification methods. For instance, production of proteins for use in screening for modulating agents may require methods that preserve specific biological activities of these proteins, whereas production of proteins for antibody generation may require structural integrity of particular epitopes. Expression of proteins to be purified for screening or antibody production may require the addition of specific tags (e.g., generation of fusion proteins). Overexpression of a KIF23 protein for assays used to assess KIF23 function, such as involvement in cell cycle regulation or hypoxic response, may require expression in eukaryotic cell lines capable of these cellular activities. Techniques for the expression, production, and purification of proteins are well known in the art; any suitable means therefore may be used (e.g., Higgins S J and Hames B D (eds.) Protein Expression: A Practical Approach, Oxford University Press Inc., New York 1999; Stanbury P F et al., Principles of Fermentation Technology, 2nd edition, Elsevier Science, New York, 1995; Doonan S (ed.) Protein Purification Protocols, Humana Press, New Jersey, 1996; Coligan J E et al, Current Protocols in Protein Science (eds.), 1999, John Wiley & Sons, New York). In particular embodiments, recombinant KIF23 is expressed in a cell line known to have defective RHO function. The recombinant cells are used in cell-based screening assay systems of the invention, as described further below.
The nucleotide sequence encoding a KIF23 polypeptide can be inserted into any appropriate expression vector. The necessary transcriptional and translational signals, including promoter/enhancer element, can derive from the native KIF23 gene and/or its flanking regions or can be heterologous. A variety of host-vector expression systems may be utilized, such as mammalian cell systems infected with virus (e.g. vaccinia virus, adenovirus, etc.); insect cell systems infected with virus (e.g. baculovirus); microorganisms such as yeast containing yeast vectors, or bacteria transformed with bacteriophage, plasmid, or cosmid DNA. An isolated host cell strain that modulates the expression of, modifies, and/or specifically processes the gene product may be used.
To detect expression of the KIF23 gene product, the expression vector can comprise a promoter operably linked to a KIF23 gene nucleic acid, one or more origins of replication, and, one or more selectable markers (e.g. thymidine kinase activity, resistance to antibiotics, etc.). Alternatively, recombinant expression vectors can be identified by assaying for the expression of the KIF23 gene product based on the physical or functional properties of the KIF23 protein in in vitro assay systems (e.g. immunoassays).
The KIF23 protein, fragment, or derivative may be optionally expressed as a fusion, or chimeric protein product (i.e. it is joined via a peptide bond to a heterologous protein sequence of a different protein), for example to facilitate purification or detection. A chimeric product can be made by ligating the appropriate nucleic acid sequences encoding the desired amino acid sequences to each other using standard methods and expressing the chimeric product. A chimeric product may also be made by protein synthetic techniques, e.g. by use of a peptide synthesizer (Hunkapiller et al., Nature (1984) 310:105-111).
Once a recombinant cell that expresses the KIF23 gene sequence is identified, the gene product can be isolated and purified using standard methods (e.g. ion exchange, affinity, and gel exclusion chromatography; centrifugation; differential solubility; electrophoresis). Alternatively, native KIF23 proteins can be purified from natural sources, by standard methods (e.g. immunoaffinity purification). Once a protein is obtained, it may be quantified and its activity measured by appropriate methods, such as immunoassay, bioassay, or other measurements of physical properties, such as crystallography.
The methods of this invention may also use cells that have been engineered for altered expression (mis-expression) of KIF23 or other genes associated with the RHO pathway. As used herein, mis-expression encompasses ectopic expression, over-expression, under-expression, and non-expression (e.g. by gene knock-out or blocking expression that would otherwise normally occur).
Genetically Modified Animals
Animal models that have been genetically modified to alter KIF23 expression may be used in in vivo assays to test for activity of a candidate RHO modulating agent, or to further assess the role of KIF23 in a RHO pathway process such as apoptosis or cell proliferation. Preferably, the altered KIF23 expression results in a detectable phenotype, such as decreased or increased levels of cell proliferation, angiogenesis, or apoptosis compared to control animals having normal KIF23 expression. The genetically modified animal may additionally have altered RHO expression (e.g. RHO knockout). Preferred genetically modified animals are mammals such as primates, rodents (preferably mice or rats), among others. Preferred non-mammalian species include zebrafish, C. elegans, and Drosophila. Preferred genetically modified animals are transgenic animals having a heterologous nucleic acid sequence present as an extrachromosomal element in a portion of its cells, i.e. mosaic animals (see, for example, techniques described by Jakobovits, 1994, Curr. Biol. 4:761-763.) or stably integrated into its germ line DNA (i.e., in the genomic sequence of most or all of its cells). Heterologous nucleic acid is introduced into the germ line of such transgenic animals by genetic manipulation of, for example, embryos or embryonic stem cells of the host animal.
Methods of making transgenic animals are well-known in the art (for transgenic mice see Brinster et al., Proc. Nat. Acad. Sci. USA 82: 4438-4442 (1985), U.S. Pat. Nos. 4,736,866 and 4,870,009, both by Leder et al., U.S. Pat. No. 4,873,191 by Wagner et al., and Hogan, B., Manipulating the Mouse Embryo, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., (1986); for particle bombardment see U.S. Pat. No. 4,945,050, by Sandford et al.; for transgenic Drosophila see Rubin and Spradling, Science (1982) 218:348-53 and U.S. Pat. No. 4,670,388; for transgenic insects see Berghammer A. J. et al., A Universal Marker for Transgenic Insects (1999) Nature 402:370-371; for transgenic Zebrafish see Lin S., Transgenic Zebrafish, Methods Mol Biol. (2000); 136:375-3830); for microinjection procedures for fish, amphibian eggs and birds see Houdebine and Chourrout, Experientia (1991) 47:897-905; for transgenic rats see Hammer et al., Cell (1990) 63:1099-1112; and for culturing of embryonic stem (ES) cells and the subsequent production of transgenic animals by the introduction of DNA into ES cells using methods such as electroporation, calcium phosphate/DNA precipitation and direct injection see, e.g., Teratocarcinomas and Embryonic Stem Cells, A Practical Approach, E. J. Robertson, ed., IRL Press (1987)). Clones of the nonhuman transgenic animals can be produced according to available methods (see Wilmut, I. et al. (1997) Nature 385:810-813; and PCT International Publication Nos. WO 97/07668 and WO 97/07669).
In one embodiment, the transgenic animal is a “knock-out” animal having a heterozygous or homozygous alteration in the sequence of an endogenous KIF23 gene that results in a decrease of KIF23 function, preferably such that KIF23 expression is undetectable or insignificant. Knock-out animals are typically generated by homologous recombination with a vector comprising a transgene having at least a portion of the gene to be knocked out. Typically a deletion, addition or substitution has been introduced into the transgene to functionally disrupt it. The transgene can be a human gene (e.g., from a human genomic clone) but more preferably is an ortholog of the human gene derived from the transgenic host species. For example, a mouse KIF23 gene is used to construct a homologous recombination vector suitable for altering an endogenous KIF23 gene in the mouse genome. Detailed methodologies for homologous recombination in mice are available (see Capecchi, Science (1989) 244:1288-1292; Joyner et al., Nature (1989) 338:153-156). Procedures for the production of non-rodent transgenic mammals and other animals are also available (Houdebine and Chourrout, supra; Pursel et al., Science (1989) 244:1281-1288; Simms et al., Bio/Technology (1988) 6:179-183). In a preferred embodiment, knock-out animals, such as mice harboring a knockout of a specific gene, may be used to produce antibodies against the human counterpart of the gene that has been knocked out (Claesson M H et al., (1994) Scan J Immunol 40:257-264; Declerck P J et al., (1995) J Biol Chem. 270:8397-400).
In another embodiment, the transgenic animal is a “knock-in” animal having an alteration in its genome that results in altered expression (e.g., increased (including ectopic) or decreased expression) of the KIF23 gene, e.g., by introduction of additional copies of KIF23, or by operatively inserting a regulatory sequence that provides for altered expression of an endogenous copy of the KIF23 gene. Such regulatory sequences include inducible, tissue-specific, and constitutive promoters and enhancer elements. The knock-in can be homozygous or heterozygous.
Transgenic nonhuman animals can also be produced that contain selected systems allowing for regulated expression of the transgene. One example of such a system that may be produced is the cre/loxP recombinase system of bacteriophage P1 (Lakso et al., PNAS (1992) 89:6232-6236; U.S. Pat. No. 4,959,317). If a cre/loxP recombinase system is used to regulate expression of the transgene, animals containing transgenes encoding both the Cre recombinase and a selected protein are required. Such animals can be provided through the construction of “double” transgenic animals, e.g., by mating two transgenic animals, one containing a transgene encoding a selected protein and the other containing a transgene encoding a recombinase. Another example of a recombinase system is the FLP recombinase system of Saccharomyces cerevisiae (O'Gorman et al. (1991) Science 251:1351-1355; U.S. Pat. No. 5,654,182). In a preferred embodiment, both Cre-LoxP and Flp-Frt are used in the same system to regulate expression of the transgene, and for sequential deletion of vector sequences in the same cell (Sun X et al (2000) Nat Genet 25:83-6).
The genetically modified animals can be used in genetic studies to further elucidate the RHO pathway, as animal models of disease and disorders implicating defective RHO function, and for in vivo testing of candidate therapeutic agents, such as those identified in screens described below. The candidate therapeutic agents are administered to a genetically modified animal having altered KIF23 function and phenotypic changes are compared with appropriate control animals such as genetically modified animals that receive placebo treatment, and/or animals with unaltered KIF23 expression that receive candidate therapeutic agent.
In addition to the above-described genetically modified animals having altered KIF23 function, animal models having defective RHO function (and otherwise normal KIF23 function), can be used in the methods of the present invention. For example, a RHO knockout mouse can be used to assess, in vivo, the activity of a candidate RHO modulating agent identified in one of the in vitro assays described below. Preferably, the candidate RHO modulating agent when administered to a model system with cells defective in RHO function, produces a detectable phenotypic change in the model system indicating that the RHO function is restored, i.e., the cells exhibit normal cell cycle progression.
Modulating Agents
The invention provides methods to identify agents that interact with and/or modulate the function of KIF23 and/or the RHO pathway. Modulating agents identified by the methods are also part of the invention. Such agents are useful in a variety of diagnostic and therapeutic applications associated with the RHO pathway, as well as in further analysis of the KIF23 protein and its contribution to the RHO pathway. Accordingly, the invention also provides methods for modulating the RHO pathway comprising the step of specifically modulating KIF23 activity by administering an KIF23-interacting or -modulating agent.
As used herein, a “KIF23-modulating agent” is any agent that modulates KIF23 function, for example, an agent that interacts with KIF23 to inhibit or enhance KIF23 activity or otherwise affect normal KIF23 function. KIF23 function can be affected at any level, including transcription, protein expression, protein localization, and cellular or extra-cellular activity. In a preferred embodiment, the KIF23-modulating agent specifically modulates the function of KIF23. The phrases “specific modulating agent”, “specifically modulates”, etc., are used herein to refer to modulating agents that directly bind to the KIF23 polypeptide or nucleic acid, and preferably inhibit, enhance, or otherwise alter, the function of KIF23. These phrases also encompass modulating agents that alter the interaction of KIF23 with a binding partner, substrate, or cofactor (e.g. by binding to a binding partner of KIF23, or to a protein/binding partner complex, and altering KIF23 function). In a further preferred embodiment, the KIF23-modulating agent is a modulator of the RHO pathway (e.g. it restores and/or upregulates RHO function) and thus is also a RHO-modulating agent.
Preferred KIF23-modulating agents include small molecule compounds; KIF23-interacting proteins, including antibodies and other biotherapeutics; and nucleic acid modulators such as antisense and RNA inhibitors. The modulating agents may be formulated in pharmaceutical compositions, for example, as compositions that may comprise other active ingredients, as in combination therapy, and/or suitable carriers or excipients. Techniques for formulation and administration of the compounds may be found in “Remington's Pharmaceutical Sciences” Mack Publishing Co., Easton, Pa., 19th edition.
Small Molecule Modulators
Small molecules are often preferred to modulate function of proteins with enzymatic function, and/or containing protein interaction domains. Chemical agents, referred to in the art as “small molecule” compounds are typically organic, non-peptide molecules, having a molecular weight up to 10,000, preferably up to 5,000, more preferably up to 1,000, and most preferably up to 500 daltons. This class of modulators includes chemically synthesized molecules, for instance, compounds from combinatorial chemical libraries. Synthetic compounds may be rationally designed or identified based on known or inferred properties of the KIF23 protein or may be identified by screening compound libraries. Alternative appropriate modulators of this class are natural products, particularly secondary metabolites from organisms such as plants or fungi, which can also be identified by screening compound libraries for KIF23-modulating activity. Methods for generating and obtaining compounds are well known in the art (Schreiber S L, Science (2000) 151: 1964-1969; Radmann J and Gunther J, Science (2000) 151:1947-1948).
Small molecule modulators identified from screening assays, as described below, can be used as lead compounds from which candidate clinical compounds may be designed, optimized, and synthesized. Such clinical compounds may have utility in treating pathologies associated with the RHO pathway. The activity of candidate small molecule modulating agents may be improved several-fold through iterative secondary functional validation, as further described below, structure determination, and candidate modulator modification and testing. Additionally, candidate clinical compounds are generated with specific regard to clinical and pharmacological properties. For example, the reagents may be derivatized and re-screened using in vitro and in vivo assays to optimize activity and minimize toxicity for pharmaceutical development.
Protein Modulators
Specific KIF23-interacting proteins are useful in a variety of diagnostic and therapeutic applications related to the RHO pathway and related disorders, as well as in validation assays for other KIF23-modulating agents. In a preferred embodiment, KIF23-interacting proteins affect normal KIF23 function, including transcription, protein expression, protein localization, and cellular or extra-cellular activity. In another embodiment, KIF23-interacting proteins are useful in detecting and providing information about the function of KIF23 proteins, as is relevant to RHO related disorders, such as cancer (e.g., for diagnostic means).
A KIF23-interacting protein may be endogenous, i.e. one that naturally interacts genetically or biochemically with KIF23, such as a member of the KIF23 pathway that modulates KIF23 expression, localization, and/or activity. KIF23-modulators include dominant negative forms of KIF23-interacting proteins and of KIF23 proteins themselves. Yeast two-hybrid and variant screens offer preferred methods for identifying endogenous KIF23-interacting proteins (Finley, R. L. et al. (1996) in DNA Cloning-Expression Systems: A Practical Approach, eds. Glover D. & Hames B. D (Oxford University Press, Oxford, England), pp. 69-203; Fashema S F et al., Gene (2000) 250:1-14; Drees B L Curr Opin Chem Biol (1999) 3:64-70; Vidal M and Legrain P Nucleic Acids Res (1999) 27:919-29; and U.S. Pat. No. 5,928,868). Mass spectrometry is an alternative preferred method for the elucidation of protein complexes (reviewed in, e.g., Pandley A and Mann M, Nature (2000) 405:837-846; Yates J R 3rd, Trends Genet (2000) 16:5-8).
A KIF23-interacting protein may be an exogenous protein, such as an KIF23-specific antibody or a T-cell antigen receptor (see, e.g., Harlow and Lane (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory; Harlow and Lane (1999) Using antibodies: a laboratory manual. Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press). KIF23 antibodies are further discussed below.
In preferred embodiments, a KIF23-interacting protein specifically binds a KIF23 protein. In alternative preferred embodiments, a KIF23-modulating agent binds a KIF23 substrate, binding partner, or cofactor.
Antibodies
In another embodiment, the protein modulator is an KIF23 specific antibody agonist or antagonist. The antibodies have therapeutic and diagnostic utilities, and can be used in screening assays to identify KIF23 modulators. The antibodies can also be used in dissecting the portions of the KIF23 pathway responsible for various cellular responses and in the general processing and maturation of the KIF23.
Antibodies that specifically bind KIF23 polypeptides can be generated using known methods. Preferably the antibody is specific to a mammalian ortholog of KIF23 polypeptide, and more preferably, to human KIF23. Antibodies may be polyclonal, monoclonal (mAbs), humanized or chimeric antibodies, single chain antibodies, Fab fragments, F(ab′).sub.2 fragments, fragments produced by a FAb expression library, anti-idiotypic (anti-Id) antibodies, and epitope-binding fragments of any of the above. Epitopes of KIF23 which are particularly antigenic can be selected, for example, by routine screening of KIF23 polypeptides for antigenicity or by applying a theoretical method for selecting antigenic regions of a protein (Hopp and Wood (1981), Proc. Nati. Acad. Sci. U.S.A. 78:3824-28; Hopp and Wood, (1983) Mol. Immunol. 20:483-89; Sutcliffe et al., (1983) Science 219:660-66) to the amino acid sequence of an KIF23. Monoclonal antibodies with affinities of 108 M−1 preferably 109 M−1 to 1010 M−1, or stronger can be made by standard procedures as described (Harlow and Lane, supra; Goding (1986) Monoclonal Antibodies: Principles and Practice (2d ed) Academic Press, New York; and U.S. Pat. Nos. 4,381,292; 4,451,570; and 4,618,577). Antibodies may be generated against crude cell extracts of KIF23 or substantially purified fragments thereof. If KIF23 fragments are used, they preferably comprise at least 10, and more preferably, at least 20 contiguous amino acids of an KIF23 protein. In a particular embodiment, KIF23-specific antigens and/or immunogens are coupled to carrier proteins that stimulate the immune response. For example, the subject polypeptides are covalently coupled to the keyhole limpet hemocyanin (KLH) carrier, and the conjugate is emulsified in Freund's complete adjuvant, which enhances the immune response. An appropriate immune system such as a laboratory rabbit or mouse is immunized according to conventional protocols.
The presence of KIF23-specific antibodies is assayed by an appropriate assay such as a solid phase enzyme-linked immunosorbant assay (ELISA) using immobilized corresponding KIF23 polypeptides. Other assays, such as radioimmunoassays or fluorescent assays might also be used.
Chimeric antibodies specific to KIF23 polypeptides can be made that contain different portions from different animal species. For instance, a human immunoglobulin constant region may be linked to a variable region of a murine mAb, such that the antibody derives its biological activity from the human antibody, and its binding specificity from the murine fragment. Chimeric antibodies are produced by splicing together genes that encode the appropriate regions from each species (Morrison et al., Proc. Natl. Acad. Sci. (1984) 81:6851-6855; Neuberger et al., Nature (1984) 312:604-608; Takeda et al., Nature (1985) 31:452-454). Humanized antibodies, which are a form of chimeric antibodies, can be generated by grafting complementary-determining regions (CDRs) (Carlos, T. M., J. M. Harlan. 1994. Blood 84:2068-2101) of mouse antibodies into a background of human framework regions and constant regions by recombinant DNA technology (Riechmann L M, et al., 1988 Nature 323: 323-327). Humanized antibodies contain 10% murine sequences and ˜90% human sequences, and thus further reduce or eliminate immunogenicity, while retaining the antibody specificities (Co M S, and Queen C. 1991 Nature 351: 501-501; Morrison S L. 1992 Ann. Rev. Immun. 10:239-265). Humanized antibodies and methods of their production are well-known in the art (U.S. Pat. Nos. 5,530,101, 5,585,089, 5,693,762, and 6,180,370).
KIF23-specific single chain antibodies which are recombinant, single chain polypeptides formed by linking the heavy and light chain fragments of the Fv regions via an amino acid bridge, can be produced by methods known in the art (U.S. Pat. No. 4,946,778; Bird, Science (1988) 242:423-426; Huston et al., Proc. Natl. Acad. Sci. USA (1988) 85:5879-5883; and Ward et al., Nature (1989) 334:544-546).
Other suitable techniques for antibody production involve in vitro exposure of lymphocytes to the antigenic polypeptides or alternatively to selection of libraries of antibodies in phage or similar vectors (Huse et al., Science (1989) 246:1275-1281). As used herein, T-cell antigen receptors are included within the scope of antibody modulators (Harlow and Lane, 1988, supra).
The polypeptides and antibodies of the present invention may be used with or without modification. Frequently, antibodies will be labeled by joining, either covalently or non-covalently, a substance that provides for a detectable signal, or that is toxic to cells that express the targeted protein (Menard S, et al., Int J. Biol Markers (1989) 4:131-134). A wide variety of labels and conjugation techniques are known and are reported extensively in both the scientific and patent literature. Suitable labels include radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent moieties, fluorescent emitting lanthanide metals, chemiluminescent moieties, bioluminescent moieties, magnetic particles, and the like (U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; and 4,366,241). Also, recombinant immunoglobulins may be produced (U.S. Pat. No. 4,816,567). Antibodies to cytoplasmic polypeptides may be delivered and reach their targets by conjugation with membrane-penetrating toxin proteins (U.S. Pat. No. 6,086,900).
When used therapeutically in a patient, the antibodies of the subject invention are typically administered parenterally, when possible at the target site, or intravenously. The therapeutically effective dose and dosage regimen is determined by clinical studies. Typically, the amount of antibody administered is in the range of about 0.1 mg/kg-to about 10 mg/kg of patient weight. For parenteral administration, the antibodies are formulated in a unit dosage injectable form (e.g., solution, suspension, emulsion) in association with a pharmaceutically acceptable vehicle. Such vehicles are inherently nontoxic and non-therapeutic. Examples are water, saline, Ringer's solution, dextrose solution, and 5% human serum albumin. Nonaqueous vehicles such as fixed oils, ethyl oleate, or liposome carriers may also be used. The vehicle may contain minor amounts of additives, such as buffers and preservatives, which enhance isotonicity and chemical stability or otherwise enhance therapeutic potential. The antibodies' concentrations in such vehicles are typically in the range of about 1 mg/ml to about 10 mg/ml. Immunotherapeutic methods are further described in the literature (U.S. Pat. No. 5,859,206; WO0073469).
Specific Biotherapeutics
In a preferred embodiment, a KIF23-interacting protein may have biotherapeutic applications. Biotherapeutic agents formulated in pharmaceutically acceptable carriers and dosages may be used to activate or inhibit signal transduction pathways. This modulation may be accomplished by binding a ligand, thus inhibiting the activity of the pathway; or by binding a receptor, either to inhibit activation of, or to activate, the receptor. Alternatively, the biotherapeutic may itself be a ligand capable of activating or inhibiting a receptor. Biotherapeutic agents and methods of producing them are described in detail in U.S. Pat. No. 6,146,628.
When the KIF23 is a ligand, it may be used as a biotherapeutic agent to activate or inhibit its natural receptor. Alternatively, antibodies against KIF23, as described in the previous section, may be used as biotherapeutic agents.
When the KIF23 is a receptor, its ligand(s), antibodies to the ligand(s) or the KIF23 itself may be used as biotherapeutics to modulate the activity of KIF23 in the RHO pathway.
Nucleic Acid Modulators
Other preferred KIF23-modulating agents comprise nucleic acid molecules, such as antisense oligomers or double stranded RNA (dsRNA), which generally inhibit KIF23 activity. Preferred nucleic acid modulators interfere with the function of the KIF23 nucleic acid such as DNA replication, transcription, translocation of the KIF23 RNA to the site of protein translation, translation of protein from the KIF23 RNA, splicing of the KIF23 RNA to yield one or more mRNA species, or catalytic activity which may be engaged in or facilitated by the KIF23 RNA.
In one embodiment, the antisense oligomer is an oligonucleotide that is sufficiently complementary to a KIF23 mRNA to bind to and prevent translation, preferably by binding to the 5′ untranslated region. KIF23-specific antisense oligonucleotides, preferably range from at least 6 to about 200 nucleotides. In some embodiments the oligonucleotide is preferably at least 10, 15, or 20 nucleotides in length. In other embodiments, the oligonucleotide is preferably less than 50, 40, or 30 nucleotides in length. The oligonucleotide can be DNA or RNA or a chimeric mixture or derivatives or modified versions thereof, single-stranded or double-stranded. The oligonucleotide can be modified at the base moiety, sugar moiety, or phosphate backbone. The oligonucleotide may include other appending groups such as peptides, agents that facilitate transport across the cell membrane, hybridization-triggered cleavage agents, and intercalating agents.
In another embodiment, the antisense oligomer is a phosphothioate morpholino oligomer (PMO). PMOs are assembled from four different morpholino subunits, each of which contain one of four genetic bases (A, C, G, or T) linked to a six-membered morpholine ring. Polymers of these subunits are joined by non-ionic phosphodiamidate intersubunit linkages. Details of how to make and use PMOs and other antisense oligomers are well known in the art (e.g. see WO99/18193; Probst J C, Antisense Oligodeoxynucleotide and Ribozyme Design, Methods. (2000) 22(3):271-281; Summerton J, and Weller D. 1997 Antisense Nucleic Acid Drug Dev.: 7:187-95; U.S. Pat. No. 5,235,033; and U.S. Pat. No. 5,378,841).
Alternative preferred KIF23 nucleic acid modulators are double-stranded RNA species mediating RNA interference (RNAi). RNAi is the process of sequence-specific, post-transcriptional gene silencing in animals and plants, initiated by double-stranded RNA (dsRNA) that is homologous in sequence to the silenced gene. Methods relating to the use of RNAi to silence genes in C. elegans, Drosophila, plants, and humans are known in the art (Fire A, et al., 1998 Nature 391:806-811; Fire, A. Trends Genet. 15, 358-363 (1999); Sharp, P. A. RNA interference 2001. Genes Dev. 15, 485-490 (2001); Hammond, S. M., et al., Nature Rev. Genet. 2, 110-1119 (2001); Tuschl, T. Chem. Biochem. 2, 239-245 (2001); Hamilton, A. et al., Science 286, 950-952 (1999); Hammond, S. M., et al., Nature 404, 293-296 (2000); Zamore, P. D., et al., Cell 101, 25-33 (2000); Bernstein, E., et al., Nature 409, 363-366 (2001); Elbashir, S. M., et al., Genes Dev. 15, 188-200 (2001); WO0129058; WO9932619; Elbashir S M, et al., 2001 Nature 411:494-498; Novina C D and Sharp P. 2004 Nature 430:161-164; Soutschek J et al 2004 Nature 432:173-178; Hsieh A C et al. (2004) NAR 32(3):893-901).
Nucleic acid modulators are commonly used as research reagents, diagnostics, and therapeutics. For example, antisense oligonucleotides, which are able to inhibit gene expression with exquisite specificity, are often used to elucidate the function of particular genes (see, for example, U.S. Pat. No. 6,165,790). Nucleic acid modulators are also used, for example, to distinguish between functions of various members of a biological pathway. For example, antisense oligomers have been employed as therapeutic moieties in the treatment of disease states in animals and man and have been demonstrated in numerous clinical trials to be safe and effective (Milligan J F, et al, Current Concepts in Antisense Drug Design, J Med Chem. (1993) 36:1923-1937: Tonkinson J L et al., Antisense Oligodeoxynucleotides as Clinical Therapeutic Agents, Cancer Invest. (1996) 14:54-65). Accordingly, in one aspect of the invention, a KIF23-specific nucleic acid modulator is used in an assay to further elucidate the role of KIF23 in the RHO pathway, and/or its relationship to other members of the pathway. In another aspect of the invention, a KIF23-specific antisense oligomer is used as a therapeutic agent for treatment of RHO-related disease states.
Assay Systems
The invention provides assay systems and screening methods for identifying specific modulators of KIF23 activity. As used herein, an “assay system” encompasses all the components required for performing and analyzing results of an assay that detects and/or measures a particular event. In general, primary assays are used to identify or confirm a modulator's specific biochemical or molecular effect with respect to the KIF23 nucleic acid or protein. In general, secondary assays further assess the activity of a KIF23 modulating agent identified by a primary assay and may confirm that the modulating agent affects KIF23 in a manner relevant to the RHO pathway. In some cases, KIF23 modulators will be directly tested in a secondary assay.
In a preferred embodiment, the screening method comprises contacting a suitable assay system comprising a KIF23 polypeptide or nucleic acid with a candidate agent under conditions whereby, but for the presence of the agent, the system provides a reference activity (e.g. binding activity), which is based on the particular molecular event the screening method detects. A statistically significant difference between the agent-biased activity and the reference activity indicates that the candidate agent modulates KIF23 activity, and hence the RHO pathway. The KIF23 polypeptide or nucleic acid used in the assay may comprise any of the nucleic acids or polypeptides described above.
Primary Assays
The type of modulator tested generally determines the type of primary assay.
Primary Assays for Small Molecule Modulators
For small molecule modulators, screening assays are used to identify candidate modulators. Screening assays may be cell-based or may use a cell-free system that recreates or retains the relevant biochemical reaction of the target protein (reviewed in Sittampalam G S et al., Curr Opin Chem Biol (1997) 1:384-91 and accompanying references). As used herein the term “cell-based” refers to assays using live cells, dead cells, or a particular cellular fraction, such as a membrane, endoplasmic reticulum, or mitochondrial fraction. The term “cell free” encompasses assays using substantially purified protein (either endogenous or recombinantly produced), partially purified or crude cellular extracts. Screening assays may detect a variety of molecular events, including protein-DNA interactions, protein-protein interactions (e.g., receptor-ligand binding), transcriptional activity (e.g., using a reporter gene), enzymatic activity (e.g., via a property of the substrate), activity of second messengers, immunogenicity and changes in cellular morphology or other cellular characteristics. Appropriate screening assays may use a wide range of detection methods including fluorescent, radioactive, colorimetric, spectrophotometric, and amperometric methods, to provide a read-out for the particular molecular event detected.
Cell-based screening assays usually require systems for recombinant expression of KIF23 and any auxiliary proteins demanded by the particular assay. Appropriate methods for generating recombinant proteins produce sufficient quantities of proteins that retain their relevant biological activities and are of sufficient purity to optimize activity and assure assay reproducibility. Yeast two-hybrid and variant screens, and mass spectrometry provide preferred methods for determining protein-protein interactions and elucidation of protein complexes. In certain applications, when KIF23-interacting proteins are used in screens to identify small molecule modulators, the binding specificity of the interacting protein to the KIF23 protein may be assayed by various known methods such as substrate processing (e.g. ability of the candidate KIF23-specific binding agents to function as negative effectors in KIF23-expressing cells), binding equilibrium constants (usually at least about 107 M−1, preferably at least about 108 M−1, more preferably at least about 109 M−1), and immunogenicity (e.g. ability to elicit KIF23 specific antibody in a heterologous host such as a mouse, rat, goat or rabbit). For enzymes and receptors, binding may be assayed by, respectively, substrate and ligand processing.
The screening assay may measure a candidate agent's ability to specifically bind to or modulate activity of a KIF23 polypeptide, a fusion protein thereof, or to cells or membranes bearing the polypeptide or fusion protein. The KIF23 polypeptide can be full length or a fragment thereof that retains functional KIF23 activity. The KIF23 polypeptide may be fused to another polypeptide, such as a peptide tag for detection or anchoring, or to another tag. The KIF23 polypeptide is preferably human KIF23, or is an ortholog or derivative thereof as described above. In a preferred embodiment, the screening assay detects candidate agent-based modulation of KIF23 interaction with a binding target, such as an endogenous or exogenous protein or other substrate that has KIF23-specific binding activity, and can be used to assess normal KIF23 gene function.
Suitable assay formats that may be adapted to screen for KIF23 modulators are known in the art. Preferred screening assays are high throughput or ultra high throughput and thus provide automated, cost-effective means of screening compound libraries for lead compounds (Fernandes P B, Curr Opin Chem Biol (1998) 2:597-603; Sundberg S A, Curr Opin Biotechnol 2000, 11:47-53). In one preferred embodiment, screening assays uses fluorescence technologies, including fluorescence polarization, time-resolved fluorescence, and fluorescence resonance energy transfer. These systems offer means to monitor protein-protein or DNA-protein interactions in which the intensity of the signal emitted from dye-labeled molecules depends upon their interactions with partner molecules (e.g., Selvin P R, Nat Struct Biol (2000) 7:730-4; Fernandes P B, supra; Hertzberg R P and Pope A J, Curr Opin Chem Biol (2000) 4:445-451).
A variety of suitable assay systems may be used to identify candidate KIF23 and RHO pathway modulators (e.g. U.S. Pat. No. 6,165,992 and U.S. Pat. No. 6,720,162 (kinase assays); U.S. Pat. Nos. 5,550,019 and 6,133,437 (apoptosis assays); WO 01/25487 (Helicase assays), U.S. Pat. No. 6,114,132 and U.S. Pat. No. 6,720,162 (phosphatase and protease assays), U.S. Pat. Nos. 5,976,782, 6,225,118 and 6,444,434 (angiogenesis assays), among others). Specific preferred assays are described in more detail below.
Protein kinases, key signal transduction proteins that may be either membrane-associated or intracellular, catalyze the transfer of gamma phosphate from adenosine triphosphate (ATP) to a serine, threonine or tyrosine residue in a protein substrate. Radioassays, which monitor the transfer from [gamma-32P or -33P]ATP, are frequently used to assay kinase activity. For instance, a scintillation assay for p56 (lck) kinase activity monitors the transfer of the gamma phosphate from [gamma-33P] ATP to a biotinylated peptide substrate. The substrate is captured on a streptavidin coated bead that transmits the signal (Beveridge M et al., J Biomol Screen (2000) 5:205-212). This assay uses the scintillation proximity assay (SPA), in which only radio-ligand bound to receptors tethered to the surface of an SPA bead are detected by the scintillant immobilized within it, allowing binding to be measured without separation of bound from free ligand. Other assays for protein kinase activity may use antibodies that specifically recognize phosphorylated substrates. For instance, the kinase receptor activation (KIRA) assay measures receptor tyrosine kinase activity by ligand stimulating the intact receptor in cultured cells, then capturing solubilized receptor with specific antibodies and quantifying phosphorylation via phosphotyrosine ELISA (Sadick M D, Dev Biol Stand (1999) 97:121-133). Another example of antibody based assays for protein kinase activity is TRF (time-resolved fluorometry). This method utilizes europium chelate-labeled anti-phosphotyrosine antibodies to detect phosphate transfer to a polymeric substrate coated onto microtiter plate wells. The amount of phosphorylation is then detected using time-resolved, dissociation-enhanced fluorescence (Braunwalder A F, et al., Anal Biochem 1996 Jul. 1; 238(2): 159-64). Yet other assays for kinases involve uncoupled, pH sensitive assays that can be used for high-throughput screening of potential inhibitors or for determining substrate specificity. Since kinases catalyze the transfer of a gamma-phosphoryl group from ATP to an appropriate hydroxyl acceptor with the release of a proton, a pH sensitive assay is based on the detection of this proton using an appropriately matched buffer/indicator system (Chapman E and Wong C H (2002) Bioorg Med Chem. 10:551-5).
Protein phosophatases catalyze the removal of a gamma phosphate from a serine, threonine or tyrosine residue in a protein substrate. Since phosphatases act in opposition to kinases, appropriate assays measure the same parameters as kinase assays. In one example, the dephosphorylation of a fluorescently labeled peptide substrate allows trypsin cleavage of the substrate, which in turn renders the cleaved substrate significantly more fluorescent (Nishikata M et al., Biochem J (1999) 343:35-391). In another example, fluorescence polarization (FP), a solution-based, homogeneous technique requiring no immobilization or separation of reaction components, is used to develop high throughput screening (HTS) assays for protein phosphatases. This assay uses direct binding of the phosphatase with the target, and increasing concentrations of target-phosphatase increase the rate of dephosphorylation, leading to a change in polarization (Parker G J et al., (2000) J Biomol Screen 5:77-88).
Proteases are enzymes that cleave protein substrates at specific sites. Exemplary assays detect the alterations in the spectral properties of an artificial substrate that occur upon protease-mediated cleavage. In one example, synthetic caspase substrates containing four amino acid proteolysis recognition sequences, separating two different fluorescent tags are employed; fluorescence resonance energy transfer detects the proximity of these fluorophores, which indicates whether the substrate is cleaved (Mahajan N P et al., Chem Biol (1999) 6:401-409).
Helicases are involved in unwinding double stranded DNA and RNA. In one embodiment, an assay for DNA helicase activity detects the displacement of a radio-labeled oligonucleotide from single stranded DNA upon initiation of unwinding (Sivaraja M et al., Anal Biochem (1998) 265:22-27). An assay for RNA helicase activity uses the scintillation proximity (SPA) assay to detect the displacement of a radio-labeled oligonucleotide from single stranded RNA (Kyono K et al., Anal Biochem (1998) 257:120-126).
Peptidyl-prolyl isomerase (PPIase) proteins, which include cyclophilins, FK506 binding proteins and paravulins, catalyze the isomerization of cis-trans proline peptide bonds in oligopeptides and are thought to be essential for protein folding during protein synthesis in the cell. Spectrophotometric assays for PPIase activity can detect isomerization of labeled peptide substrates, either by direct measurement of isomer-specific absorbance, or by coupling isomerization to isomer-specific cleavage by chymotrypsin (Scholz C et al., FEBS Lett (1997) 414:69-73; Janowski B et al., Anal Biochem (1997) 252:299-307; Kullertz G et al., Clin Chem (1998) 44:502-8). Alternative assays use the scintillation proximity or fluorescence polarization assay to screen for ligands of specific PPIases (Graziani F et al., J Biolmol Screen (1999) 4:3-7; Dubowchik G M et al., Bioorg Med Chem Lett (2000) 10:559-562). Assays for 3,2-trans-enoyl-CoA isomerase activity have also been described (Binstock, J. F., and Schulz, H. (1981) Methods Enzymol. 71:403-411; Geisbrecht B V et al (1999) J Biol Chem. 274:21797-803). These assays use 3-cis-octenoyl-CoA as a substrate, and reaction progress is monitored spectrophotometrically using a coupled assay for the isomerization of 3-cis-octenoyl-CoA to 2-trans-octenoyl-CoA.
Ubiquitination is a process of attaching ubiquitin to a protein prior to the selective proteolysis of that protein in the cell. Assays based on fluorescence resonance energy transfer to screen for ubiquitination inhibitors are known in the art (Boisclair M D et al., J Biomol Screen 2000 5:319-328).
Hydrolases catalyze the hydrolysis of a substrate such as esterases, lipases, peptidases, nucleotidases, and phosphatases, among others. Enzyme activity assays may be used to measure hydrolase activity. The activity of the enzyme is determined in presence of excess substrate, by spectrophotometrically measuring the rate of appearance of reaction products. High throughput arrays and assays for hydrolases are known to those skilled in the art (Park C B and Clark D S (2002) Biotech Bioeng 78:229-235).
Kinesins are motor proteins. Assays for kinesins involve their ATPase activity, such as described in Blackburn et al (Blackburn C L, et al., (1999) J Org Chem 64:5565-5570). The ATPase assay is performed using the EnzCheck ATPase kit (Molecular Probes). The assays are performed using an Ultraspec spectrophotometer (Pharmacia), and the progress of the reaction are monitored by absorbance increase at 360 nm. Microtubules (1.7 mM final), kinesin (0.11 mM final), inhibitor (or DMSO blank at 5% final), and the EnzCheck components (purine nucleotide phosphorylase and MESG substrate) are premixed in the cuvette in a reaction buffer (40 mM PIPES pH 6.8, 5 mM paclitaxel, 1 mM EGTA, 5 mM MgCl2). The reaction is initiated by addition of MgATP (1 mM final).
High-throughput assays, such as scintillation proximity assays, for synthase enzymes involved in fatty acid synthesis are known in the art (He X et al (2000) Anal Biochem 2000 Jun. 15; 282(1):107-14).
Apoptosis assays. Apoptosis or programmed cell death is a suicide program is activated within the cell, leading to fragmentation of DNA, shrinkage of the cytoplasm, membrane changes and cell death. Apoptosis is mediated by proteolytic enzymes of the caspase family. Many of the altering parameters of a cell are measurable during apoptosis. Assays for apoptosis may be performed by terminal deoxynucleotidyl transferase-mediated digoxigenin-11-dUTP nick end labeling (TUNEL) assay. The TUNEL assay is used to measure nuclear DNA fragmentation characteristic of apoptosis (Lazebnik et al, 1994, Nature 371, 346), by following the incorporation of fluorescein-dUTP (Yonehara et al., 1989, J. Exp. Med. 169, 1747). Apoptosis may further be assayed by acridine orange staining of tissue culture cells (Lucas, R., et al., 1998, Blood 15:4730-41). Other cell-based apoptosis assays include the caspase-3/7 assay and the cell death nucleosome ELISA assay. The caspase 3/7 assay is based on the activation of the caspase cleavage activity as part of a cascade of events that occur during programmed cell death in many apoptotic pathways. In the caspase 3/7 assay (commercially available Apo-ONE™ Homogeneous Caspase-3/7 assay from Promega, cat#67790), lysis buffer and caspase substrate are mixed and added to cells. The caspase substrate becomes fluorescent when cleaved by active caspase 3/7. The nucleosome ELISA assay is a general cell death assay known to those skilled in the art, and available commercially (Roche, Cat#1774425). This assay is a quantitative sandwich-enzyme-immunoassay which uses monoclonal antibodies directed against DNA and histones respectively, thus specifically determining amount of mono- and oligonucleosomes in the cytoplasmic fraction of cell lysates. Mono and oligonucleosomes are enriched in the cytoplasm during apoptosis due to the fact that DNA fragmentation occurs several hours before the plasma membrane breaks down, allowing for accumulation in the cytoplasm. Nucleosomes are not present in the cytoplasmic fraction of cells that are not undergoing apoptosis. The Phospho-histone H2B assay is another apoptosis assay, based on phosphorylation of histone H2B as a result of apoptosis. Fluorescent dyes that are associated with phosphohistone H2B may be used to measure the increase of phosphohistone H2B as a result of apoptosis. Apoptosis assays that simultaneously measure multiple parameters associated with apoptosis have also been developed. In such assays, various cellular parameters that can be associated with antibodies or fluorescent dyes, and that mark various stages of apoptosis are labeled, and the results are measured using instruments such as Cellomics™ ArrayScan® HCS System. The measurable parameters and their markers include anti-active caspase-3 antibody which marks intermediate stage apoptosis, anti-PARP-p85 antibody (cleaved PARP) which marks late stage apoptosis, Hoechst labels which label the nucleus and are used to measure nuclear swelling as a measure of early apoptosis and nuclear condensation as a measure of late apoptosis, TOTO-3 fluorescent dye which labels DNA of dead cells with high cell membrane permeability, and anti-alpha-tubulin or F-actin labels, which assess cytoskeletal changes in cells and correlate well with TOTO-3 label. These assays may also be used for involvement of a gene in cell cycle and assessment of alterations in cell morphology.
An apoptosis assay system may comprise a cell that expresses an KIF23, and that optionally has defective RHO function (e.g. RHO is over-expressed or under-expressed relative to wild-type cells). A test agent can be added to the apoptosis assay system and changes in induction of apoptosis relative to controls where no test agent is added, identify candidate RHO modulating agents. In some embodiments of the invention, an apoptosis assay may be used as a secondary assay to test a candidate RHO modulating agents that is initially identified using a cell-free assay system. An apoptosis assay may also be used to test whether KIF23 function plays a direct role in apoptosis. For example, an apoptosis assay may be performed on cells that over- or under-express KIF23 relative to wild type cells. Differences in apoptotic response compared to wild type cells suggests that KIF23 plays a direct role in the apoptotic response. Apoptosis assays are described further in U.S. Pat. No. 6,133,437.
Cell proliferation and cell cycle assays. Cell proliferation may be assayed via bromodeoxyuridine (BRDU) incorporation. This assay identifies a cell population undergoing DNA synthesis by incorporation of BRDU into newly-synthesized DNA. Newly-synthesized DNA may then be detected using an anti-BRDU antibody (Hoshino et al., 1986, Int. J. Cancer 38, 369; Campana et al. 1988, J. Immunol. Meth. 107, 79), or by other means.
Cell proliferation is also assayed via phospho-histone H3 staining, which identifies a cell population undergoing mitosis by phosphorylation of histone H3. Phosphorylation of histone H3 at serine 10 is detected using an antibody specific to the phosphorylated form of the serine 10 residue of histone H3. (Chadlee, D. N. 1995, J. Biol. Chem 270:20098-105). Cell Proliferation may also be examined using [3H]-thymidine incorporation (Chen, J., 1996, Oncogene 13:1395-403; Jeoung, J., 1995, J. Biol. Chem. 270:18367-73). This assay allows for quantitative characterization of S-phase DNA syntheses. In this assay, cells synthesizing DNA will incorporate [3H]-thymidine into newly synthesized DNA. Incorporation can then be measured by standard techniques such as by counting of radioisotope in a scintillation counter (e.g., Beckman LS 3800 Liquid Scintillation Counter). Another proliferation assay uses the dye Alamar Blue (available from Biosource International), which fluoresces when reduced in living cells and provides an indirect measurement of cell number (Voytik-Harbin S L et al., 1998, In Vitro Cell Dev Biol Anim 34:239-46). Yet another proliferation assay, the MTS assay, is based on in vitro cytotoxicity assessment of industrial chemicals, and uses the soluble tetrazolium salt, MTS. MTS assays are commercially available, for example, the Promega CellTiter 96® AQueous Non-Radioactive Cell Proliferation Assay (Cat. #G5421).
Cell proliferation may also be assayed by colony formation in soft agar, or clonogenic survival assay (Sambrook et al., Molecular Cloning, Cold Spring Harbor (1989)). For example, cells transformed with KIF23 are seeded in soft agar plates, and colonies are measured and counted after two weeks incubation.
Cell proliferation may also be assayed by measuring ATP levels as indicator of metabolically active cells. Such assays are commercially available, for example Cell Titer-Glo™, which is a luminescent homogeneous assay available from Promega.
Involvement of a gene in the cell cycle may be assayed by flow cytometry (Gray J W et al. (1986) Int J Radiat Biol Relat Stud Phys Chem Med 49:237-55). Cells transfected with KIF23 may be stained with propidium iodide and evaluated in a flow cytometer (available from Becton Dickinson), which indicates accumulation of cells in different stages of the cell cycle.
Involvement of a gene in the cell cycle, cell movement, or cell morphology may further be assessed using the Cellomics™ ArrayScan® HCS System, as described above. For cell morphology assessments, a further measurable parameter and marker is fluorescent phopho-Cofilin. Cofilin is a gene involved downstream in the RHO pathway that is phosphorylated by LIMK. Decreased LIMK levels lead to reduction of phospho-cofilin, and reduced fluorescent phospho-cofilin in the assay. Genes whose expression pattern is consistent with that of LIMK are members of the RHO pathway. For cell motility, cells are seeded in 96 well plates, then treated with modulator of interest, such as RNAi, then transferred to collagen plates containing fluorescent microspheres. Replated cells are later fixed and stained with rhodamine-Alexa546, and motility tracks are viewed and measured using the HCS system.
Accordingly, a cell proliferation, cell movement, cell morphology, or cell cycle assay system may comprise a cell that expresses a KIF23, and that optionally has defective RHO function (e.g. RHO is over-expressed or under-expressed relative to wild-type cells). A test agent can be added to the assay system and changes in cell proliferation or cell cycle relative to controls where no test agent is added, identify candidate RHO modulating agents. In some embodiments of the invention, the cell proliferation or cell cycle assay may be used as a secondary assay to test a candidate RHO modulating agents that is initially identified using another assay system such as a cell-free assay system. A cell proliferation assay may also be used to test whether KIF23 function plays a direct role in cell proliferation or cell cycle. For example, a cell proliferation or cell cycle assay may be performed on cells that over- or under-express KIF23 relative to wild type cells. Differences in proliferation or cell cycle compared to wild type cells suggests that the KIF23 plays a direct role in cell proliferation or cell cycle.
Angiogenesis. Angiogenesis may be assayed using various human endothelial cell systems, such as umbilical vein, coronary artery, or dermal cells. Suitable assays include Alamar Blue based assays (available from Biosource International) to measure proliferation; migration assays using fluorescent molecules, such as the use of Becton Dickinson Falcon HTS FluoroBlock cell culture inserts to measure migration of cells through membranes in presence or absence of angiogenesis enhancer or suppressors; and tubule formation assays based on the formation of tubular structures by endothelial cells on Matrigel® (Becton Dickinson). Accordingly, an angiogenesis assay system may comprise a cell that expresses a KIF23, and that optionally has defective RHO function (e.g. RHO is over-expressed or under-expressed relative to wild-type cells). A test agent can be added to the angiogenesis assay system and changes in angiogenesis relative to controls where no test agent is added, identify candidate RHO modulating agents. In some embodiments of the invention, the angiogenesis assay may be used as a secondary assay to test a candidate RHO modulating agents that is initially identified using another assay system. An angiogenesis assay may also be used to test whether KIF23 function plays a direct role in cell proliferation. For example, an angiogenesis assay may be performed on cells that over- or under-express KIF23 relative to wild type cells. Differences in angiogenesis compared to wild type cells suggests that the KIF23 plays a direct role in angiogenesis. U.S. Pat. Nos. 5,976,782, 6,225,118 and 6,444,434, among others, describe various angiogenesis assays.
Hypoxic induction. The alpha subunit of the transcription factor, hypoxia inducible factor-1 (HIF-1), is upregulated in tumor cells following exposure to hypoxia in vitro. Under hypoxic conditions, HIF-1 stimulates the expression of genes known to be important in tumour cell survival, such as those encoding glyolytic enzymes and VEGF. Induction of such genes by hypoxic conditions may be assayed by growing cells transfected with KIF23 in hypoxic conditions (such as with 0.1% O2, 5% CO2, and balance N2, generated in a Napco 7001 incubator (Precision Scientific)) and normoxic conditions, followed by assessment of gene activity or expression by Taqman®. For example, a hypoxic induction assay system may comprise a cell that expresses KIF23, and that optionally has defective RHO function (e.g. RHO is over-expressed or under-expressed relative to wild-type cells). A test agent can be added to the hypoxic induction assay system and changes in hypoxic response relative to controls where no test agent is added, identify candidate RHO modulating agents. In some embodiments of the invention, the hypoxic induction assay may be used as a secondary assay to test a candidate RHO modulating agents that is initially identified using another assay system. A hypoxic induction assay may also be used to test whether KIF23 function plays a direct role in the hypoxic response. For example, a hypoxic induction assay may be performed on cells that over- or under-express KIF23 relative to wild type cells. Differences in hypoxic response compared to wild type cells suggests that the KIF23 plays a direct role in hypoxic induction.
Cell adhesion. Cell adhesion assays measure adhesion of cells to purified adhesion proteins, or adhesion of cells to each other, in presence or absence of candidate modulating agents. Cell-protein adhesion assays measure the ability of agents to modulate the adhesion of cells to purified proteins. For example, recombinant proteins are produced, diluted to 2.5 g/mL in PBS, and used to coat the wells of a microtiter plate. The wells used for negative control are not coated. Coated wells are then washed, blocked with 1% BSA, and washed again. Compounds are diluted to 2× final test concentration and added to the blocked, coated wells. Cells are then added to the wells, and the unbound cells are washed off. Retained cells are labeled directly on the plate by adding a membrane-permeable fluorescent dye, such as calcein-AM, and the signal is quantified in a fluorescent microplate reader.
Cell-cell adhesion assays measure the ability of agents to modulate binding of cell adhesion proteins with their native ligands. These assays use cells that naturally or recombinantly express the adhesion protein of choice. In an exemplary assay, cells expressing the cell adhesion protein are plated in wells of a multiwell plate. Cells expressing the ligand are labeled with a membrane-permeable fluorescent dye, such as BCECF, and allowed to adhere to the monolayers in the presence of candidate agents. Unbound cells are washed off, and bound cells are detected using a fluorescence plate reader.
High-throughput cell adhesion assays have also been described. In one such assay, small molecule ligands and peptides are bound to the surface of microscope slides using a microarray spotter, intact cells are then contacted with the slides, and unbound cells are washed off. In this assay, not only the binding specificity of the peptides and modulators against cell lines are determined, but also the functional cell signaling of attached cells using immunofluorescence techniques in situ on the microchip is measured (Falsey J R et al., Bioconjug Chem. 2001 May-June; 12(3):346-53).
For antibody modulators, appropriate primary assays test is a binding assay that tests the antibody's affinity to and specificity for the KIF23 protein. Methods for testing antibody affinity and specificity are well known in the art (Harlow and Lane, 1988, 1999, supra). The enzyme-linked immunosorbant assay (ELISA) is a preferred method for detecting KIF23-specific antibodies; others include FACS assays, radioimmunoassays, and fluorescent assays.
In some cases, screening assays described for small molecule modulators may also be used to test antibody modulators.
For nucleic acid modulators, primary assays may test the ability of the nucleic acid modulator to inhibit or enhance KIF23 gene expression, preferably mRNA expression. In general, expression analysis comprises comparing KIF23 expression in like populations of cells (e.g., two pools of cells that endogenously or recombinantly express KIF23) in the presence and absence of the nucleic acid modulator. Methods for analyzing mRNA and protein expression are well known in the art. For instance, Northern blotting, slot blotting, ribonuclease protection, quantitative RT-PCR (e.g., using the TaqMan®, PE Applied Biosystems), or microarray analysis may be used to confirm that KIF23 mRNA expression is reduced in cells treated with the nucleic acid modulator (e.g., Current Protocols in Molecular Biology (1994) Ausubel F M et al., eds., John Wiley & Sons, Inc., chapter 4; Freeman W M et al., Biotechniques (1999) 26:112-125; Kallioniemi O P, Ann Med 2001, 33:142-147; Blohm D H and Guiseppi-Elie, A Curr Opin Biotechnol 2001, 12:41-47). Protein expression may also be monitored. Proteins are most commonly detected with specific antibodies or antisera directed against either the KIF23 protein or specific peptides. A variety of means including Western blotting, ELISA, or in situ detection, are available (Harlow E and Lane D, 1988 and 1999, supra).
In some cases, screening assays described for small molecule modulators, particularly in assay systems that involve KIF23 mRNA expression, may also be used to test nucleic acid modulators.
Secondary assays may be used to further assess the activity of KIF23-modulating agent identified by any of the above methods to confirm that the modulating agent affects KIF23 in a manner relevant to the RHO pathway. As used herein, KIF23-modulating agents encompass candidate clinical compounds or other agents derived from previously identified modulating agent. Secondary assays can also be used to test the activity of a modulating agent on a particular genetic or biochemical pathway or to test the specificity of the modulating agent's interaction with KIF23.
Secondary assays generally compare like populations of cells or animals (e.g., two pools of cells or animals that endogenously or recombinantly express KIF23) in the presence and absence of the candidate modulator. In general, such assays test whether treatment of cells or animals with a candidate KIF23-modulating agent results in changes in the RHO pathway in comparison to untreated (or mock- or placebo-treated) cells or animals. Certain assays use “sensitized genetic backgrounds”, which, as used herein, describe cells or animals engineered for altered expression of genes in the RHO or interacting pathways.
Cell based assays may detect endogenous RHO pathway activity or may rely on recombinant expression of RHO pathway components. Any of the aforementioned assays may be used in this cell-based format. Candidate modulators are typically added to the cell media but may also be injected into cells or delivered by any other efficacious means.
A variety of non-human animal models of normal or defective RHO pathway may be used to test candidate KIF23 modulators. Models for defective RHO pathway typically use genetically modified animals that have been engineered to mis-express (e.g., over-express or lack expression in) genes involved in the RHO pathway. Assays generally require systemic delivery of the candidate modulators, such as by oral administration, injection, etc.
In a preferred embodiment, RHO pathway activity is assessed by monitoring neovascularization and angiogenesis. Animal models with defective and normal RHO are used to test the candidate modulator's affect on KIF23 in Matrigel® assays. Matrigel® is an extract of basement membrane proteins, and is composed primarily of laminin, collagen IV, and heparin sulfate proteoglycan. It is provided as a sterile liquid at 4° C., but rapidly forms a solid gel at 37° C. Liquid Matrigel® is mixed with various angiogenic agents, such as bFGF and VEGF, or with human tumor cells which over-express the KIF23. The mixture is then injected subcutaneously (SC) into female athymic nude mice (Taconic, Germantown, N.Y.) to support an intense vascular response. Mice with Matrigel®(pellets may be dosed via oral (PO), intraperitoneal (IP), or intravenous (IV) routes with the candidate modulator. Mice are euthanized 5-12 days post-injection, and the Matrigel® pellet is harvested for hemoglobin analysis (Sigma plasma hemoglobin kit). Hemoglobin content of the gel is found to correlate the degree of neovascularization in the gel.
In another preferred embodiment, the effect of the candidate modulator on KIF23 is assessed via tumorigenicity assays. Tumor xenograft assays are known in the art (see, e.g., OgawaK et al., 2000, Oncogene 19:6043-6052). Xenografts are typically implanted SC into female athymic mice, 6-7 week old, as single cell suspensions either from a pre-existing tumor or from in vitro culture. The tumors which express the KIF23 endogenously are injected in the flank, 1×105 to 1×107 cells per mouse in a volume of 100 μL using a 27 gauge needle. Mice are then ear tagged and tumors are measured twice weekly. Candidate modulator treatment is initiated on the day the mean tumor weight reaches 100 mg. Candidate modulator is delivered IV, SC, IP, or PO by bolus administration. Depending upon the pharmacokinetics of each unique candidate modulator, dosing can be performed multiple times per day. The tumor weight is assessed by measuring perpendicular diameters with a caliper and calculated by multiplying the measurements of diameters in two dimensions. At the end of the experiment, the excised tumors maybe utilized for biomarker identification or further analyses. For immunohistochemistry staining, xenograft tumors are fixed in 4% paraformaldehyde, 0.1 M phosphate, pH 7.2, for 6 hours at 4° C., immersed in 30% sucrose in PBS, and rapidly frozen in isopentane cooled with liquid nitrogen.
In another preferred embodiment, tumorogenicity is monitored using a hollow fiber assay, which is described in U.S. Pat. No. 5,698,413. Briefly, the method comprises implanting into a laboratory animal a biocompatible, semi-permeable encapsulation device containing target cells, treating the laboratory animal with a candidate modulating agent, and evaluating the target cells for reaction to the candidate modulator. Implanted cells are generally human cells from a pre-existing tumor or a tumor cell line. After an appropriate period of time, generally around six days, the implanted samples are harvested for evaluation of the candidate modulator. Tumorogenicity and modulator efficacy may be evaluated by assaying the quantity of viable cells present in the macrocapsule, which can be determined by tests known in the art, for example, MTT dye conversion assay, neutral red dye uptake, trypan blue staining, viable cell counts, the number of colonies formed in soft agar, the capacity of the cells to recover and replicate in vitro, etc.
In another preferred embodiment, a tumorogenicity assay use a transgenic animal, usually a mouse, carrying a dominant oncogene or tumor suppressor gene knockout under the control of tissue specific regulatory sequences; these assays are generally referred to as transgenic tumor assays. In a preferred application, tumor development in the transgenic model is well characterized or is controlled. In an exemplary model, the “RIP1-Tag2” transgene, comprising the SV40 large T-antigen oncogene under control of the insulin gene regulatory regions is expressed in pancreatic beta cells and results in islet cell carcinomas (Hanahan D, 1985, Nature 315:115-122; Parangi S et al, 1996, Proc Natl Acad Sci USA 93: 2002-2007; Bergers G et al, 1999, Science 284:808-812). An “angiogenic switch,” occurs at approximately five weeks, as normally quiescent capillaries in a subset of hyperproliferative islets become angiogenic. The RIP1-TAG2 mice die by age 14 weeks. Candidate modulators may be administered at a variety of stages, including just prior to the angiogenic switch (e.g., for a model of tumor prevention), during the growth of small tumors (e.g., for a model of intervention), or during the growth of large and/or invasive tumors (e.g., for a model of regression). Tumorogenicity and modulator efficacy can be evaluating life-span extension and/or tumor characteristics, including number of tumors, tumor size, tumor morphology, vessel density, apoptotic index, etc.
Specific KIF23-modulating agents are useful in a variety of diagnostic and therapeutic applications where disease or disease prognosis is related to defects in the RHO pathway, such as angiogenic, apoptotic, or cell proliferation disorders. Accordingly, the invention also provides methods for modulating the RHO pathway in a cell, preferably a cell pre-determined to have defective or impaired RHO function (e.g. due to overexpression, underexpression, or misexpression of RHO, or due to gene mutations), comprising the step of administering an agent to the cell that specifically modulates KIF23 activity. Preferably, the modulating agent produces a detectable phenotypic change in the cell indicating that the RHO function is restored. The phrase “function is restored”, and equivalents, as used herein, means that the desired phenotype is achieved, or is brought closer to normal compared to untreated cells. For example, with restored RHO function, cell proliferation and/or progression through cell cycle may normalize, or be brought closer to normal relative to untreated cells. The invention also provides methods for treating disorders or disease associated with impaired RHO function by administering a therapeutically effective amount of a KIF23-modulating agent that modulates the RHO pathway. The invention further provides methods for modulating KIF23 function in a cell, preferably a cell pre-determined to have defective or impaired KIF23 function, by administering a KIF23-modulating agent. Additionally, the invention provides a method for treating disorders or disease associated with impaired KIF23 function by administering a therapeutically effective amount of a KIF23-modulating agent.
The discovery that KIF23 is implicated in RHO pathway provides for a variety of methods that can be employed for the diagnostic and prognostic evaluation of diseases and disorders involving defects in the RHO pathway and for the identification of subjects having a predisposition to such diseases and disorders.
Various expression analysis methods can be used to diagnose whether KIF23 expression occurs in a particular sample, including Northern blotting, slot blotting, ribonuclease protection, quantitative RT-PCR, and microarray analysis. (e.g., Current Protocols in Molecular Biology (1994) Ausubel F M et al., eds., John Wiley & Sons, Inc., chapter 4; Freeman W M et al., Biotechniques (1999) 26:112-125; Kallioniemi O P, Ann Med 2001, 33:142-147; Blohm and Guiseppi-Elie, Curr Opin Biotechnol 2001, 12:41-47). Tissues having a disease or disorder implicating defective RHO signaling that express a KIF23, are identified as amenable to treatment with a KIF23 modulating agent. In a preferred application, the RHO defective tissue overexpresses KIF23 relative to normal tissue. For example, a Northern blot analysis of mRNA from tumor and normal cell lines, or from tumor and matching normal tissue samples from the same patient, using full or partial KIF23 cDNA sequences as probes, can determine whether particular tumors express or overexpress KIF23. Alternatively, the TaqMan® is used for quantitative RT-PCR analysis of KIF23 expression in cell lines, normal tissues and tumor samples (PE Applied Biosystems).
Various other diagnostic methods may be performed, for example, utilizing reagents such as the KIF23 oligonucleotides, and antibodies directed against KIF23, as described above for: (1) the detection of the presence of KIF23 gene mutations, or the detection of either over- or under-expression of KIF23 mRNA relative to the non-disorder state; (2) the detection of either an over- or an under-abundance of KIF23 gene product relative to the non-disorder state; and (3) the detection of perturbations or abnormalities in the signal transduction pathway mediated by KIF23.
Kits for detecting expression of KIF23 in various samples, comprising at least one antibody specific to KIF23, all reagents and/or devices suitable for the detection of antibodies, the immobilization of antibodies, and the like, and instructions for using such kits in diagnosis or therapy are also provided.
Thus, in a specific embodiment, the invention is drawn to a method for diagnosing a disease or disorder in a patient that is associated with alterations in KIF23 expression, the method comprising: a) obtaining a biological sample from the patient; b) contacting the sample with a probe for KIF23 expression; c) comparing results from step (b) with a control; and d) determining whether step (c) indicates a likelihood of the disease or disorder. Preferably, the disease is cancer, most preferably a cancer as shown in TABLE 2. The probe may be either DNA or protein, including an antibody.
The following experimental section and examples are offered by way of illustration and not by way of limitation.
I. C. elegans RHO Screen
A C. elegans genetic screen was designed which employed RNAi of specific genes to identify genetic modifiers of Rho pathway function. The Ect2-encoding gene, let-21, was chosen a genetic entry point for Rho pathway signaling since a weak reduction of mutant was available that shows phenotypic abnormalities similar to those of rho-1 (RNAi) animals. Like rho-1 (RNAi), the weak let-21 allele, oz93, results in a sterile germline phenotype characterized by several pathway-diagnostic defects, including defective cytokinesis and nuclear/cytoplasmic partitioning, and a meiotic cell-cycle defect that prevents germ cells from proceeding past the pachytene-stage of meiotic prophase. These phenotypes are also shared by the stronger let-21 mutant, e1778. However, the strong mutant also confers two somatic phenotypes not seen in the weak Ect2 mutant—a protruding vulva (Pv1) phenotype and an Uncoordinated (Unc) movement phenotype. As the basis for the genetic screen, we exploited the genetic principle that weak reduction-of-function mutants in a given biological pathway often show genetic synergy with perturbations elsewhere in the pathway. The weak let-21 (oz93) mutant shows this behavior as demonstrated by combinations of it with certain other known Rho pathway components. For example, the combination of let-21 (oz93) with rho-1 (RNAi) results in Pv1 and Unc phenotypes (similar to the strong let-21 mutant) that are much more penetrant than observed with let-21 (oz93) or rho-1 (RNAi) alone. A similar genetic synergy is also observed with other genes whose mammalian counterparts function upstream or downstream of rho-1. These include genes for nematode orthologues of MGCRacGAP (cyk-4 gene), Rho kinase (let-502); nonmuscle myosin heavy chain II (nmy-2), myosin light chain (MLC-2) and a formin protein (cyk-1). Based on these genetic interaction phenotypes, we postulated that a large scale enhancer screen using the let-21 (oz93) mutant should identity additional novel genes whose normal function is to promote Rho/Ect2 pathway signaling
The let-21/Ect2 enhancer screen combined the use of a genetically-optimized let-21 (oz93) strain together with a library of double-stranded RNAs (dsRNA) made to approximately 3100 C. elegans genes. Genes represented in the RNA library were selected predominantly based on their containing enzymatic domains as determined by such informatic methods as PFAM searches and annotations in databases of C. elegans genomic information (e.g., WormBase, Worm Proteosome Database). The let-21 (oz93) strain is a genetically-balanced strain of the genotype let-21 (oz93) sqt-1 (sc13)/mnC1; eri-1(mg366). This strain segregates both let-21 sqt-1 double homozygotes, which are recognizable by their Roller behavioral phenotype (conferred by the sqt-1 mutation), and let-21 sqt-1/++ heterozygotes, which are non-Roller. The mnC1 chromosomal inversion serves to prevent recombination, while the eri-1 mutation confers enhanced sensitivity to RNAi (Kennedy S et al (2004) Nature 427(6975):645-649).
In the genetic screen, a mixture of let-21 (oz93) homozygotes and heterozygotes were collected at L1 diapause, incubated 24 hr at 200° C. with a 3.5× volume of dsRNA corresponding to individual genes, and then plated onto nematode growth plates. Three to four days later, the plates were scored for the presence of worms exhibiting Pv1 and/or Unc phenotypes; where appropriate the percentage of worms expressing each phenotype was determined separately for both let-21 homozygotes and heterozygotes. Genes were scored as showing genetic synergy with let-21 if let-21 (oz93) homozygotes expressed the Pv1 and/or Unc phenotypes at a significantly higher frequency than did let-21 (oz93) heterozygotes. Statistical significance (p<0.05) for genetic synergy was determined by a modified Test Stat function (where the Test stat=[(fraction mutant let-21 (oz93); geneX(RNAi) animals)−(fraction mutant let-21(oz93)+fraction mutant geneX(RNAi) animals)]/square root (total invariance); p was determined as the Chi Square distribution of the Test Stat value squared divided by 2).
The screen identified genes that showed significant genetic synergy with let-21 (oz93) for either the Pv1 phenotype alone or for both the Pv1 and Unc phenotypes. A partial validation of the screen was provided by observation that 20 modifiers from the screen are homologous to mammalian genes whose products have been linked in published literature to signaling pathways involving Rho, Rac, or CDC42. As an additional method of pathway validation, two genetic secondary assays were designed and used to test the modifiers. In one, a constitutively activated rho-1 transgene, containing a glycine-to-valine substitution at position 12 was constructed and expressed in worms under control the lin-31 promoter, which drives expression in the developing vulval cells. A strain containing this transgene integrated into the genome expressed a Multi-vulva phenotype that was approximately 80% penetrant (in a rrf-3 RNAi hypersensitivity background). A subset of 12 modifiers from the screen partially suppressed the activated rho-1 phenotype, indicating that at a genetic level these genes function downstream of (or in parallel with) rho-1. The second genetic validation assay utilized a temperature-sensitive mutant of myosin light phosphate encoding gene, mel-11. This mutant, mel-11(it26) displayed an embryonic lethal phenotype that was strongly suppressed by RNAi of the C. elegans orthologues of rho kinase (let-502), myosin heavy chain (nmy-1) or myosin light chain (mlc-4), but not by RNAi of Rho (rho-1) or Ect2 (let-21). Based on this differential sensitively, suppression of mel-11(it26) appears to identify genes that act downstream of rho-1 in a Rock/nonmuscle myosin signaling pathway.
BLAST analysis (Altschul et al., supra) was employed to identify orthologs of C. elegans modifiers. The columns “MRHO symbol”, and “MRHO name aliases” provide a symbol and the known name abbreviations for the Targets, where available, from Genbank. “MRHO RefSeq_NA or GI_NA”, “MRHO GI_AA”, “MRHO NAME”, and “MRHO Description” provide the reference DNA sequences for the MRHO s as available from National Center for Biology Information (NCBI), MRHO protein Genbank identifier number (GI#), MRHO name, and MRHO description, all available from Genbank, respectively. The length of each amino acid is in the “MRHO Protein Length” column.
Names and Protein sequences of C. elegans modifiers of RHO from screen (Example I), are represented in the “Modifier Name” and “Modifier GI_AA” column by GI#, respectively.
A purified or partially purified KIF23 is diluted in a suitable reaction buffer, e.g., 50 mM Hepes, pH 7.5, containing magnesium chloride or manganese chloride (1-20 mM) and a peptide or polypeptide substrate, such as myelin basic protein or casein (1-10 μg/ml). The final concentration of the kinase is 1-20 nM. The enzyme reaction is conducted in microtiter plates to facilitate optimization of reaction conditions by increasing assay throughput. A 96-well microtiter plate is employed using a final volume 30-100 μl. The reaction is initiated by the addition of 33P-gamma-ATP (0.5 μCi/ml) and incubated for 0.5 to 3 hours at room temperature. Negative controls are provided by the addition of EDTA, which chelates the divalent cation (Mg2+ or Mn2+) required for enzymatic activity. Following the incubation, the enzyme reaction is quenched using EDTA. Samples of the reaction are transferred to a 96-well glass fiber filter plate (MultiScreen, Millipore). The filters are subsequently washed with phosphate-buffered saline, dilute phosphoric acid (0.5%) or other suitable medium to remove excess radiolabeled ATP. Scintillation cocktail is added to the filter plate and the incorporated radioactivity is quantitated by scintillation counting (Wallac/Perkin Elmer). Activity is defined by the amount of radioactivity detected following subtraction of the negative control reaction value (EDTA quench).
All cell lines used in the following experiments are NCI (National Cancer Institute) lines, and are available from ATCC (American Type Culture Collection, Manassas, Va. 20110-2209). Normal and tumor tissues were obtained from Impath, UC Davis, Clontech, Stratagene, Ardais, Genome Collaborative, and Ambion.
TaqMan® analysis was used to assess expression levels of the disclosed genes in various samples.
RNA was extracted from each tissue sample using Qiagen (Valencia, Calif.) RNeasy kits, following manufacturer's protocols, to a final concentration of 50 ng/μl. Single stranded cDNA was then synthesized by reverse transcribing the RNA samples using random hexamers and 500 ng of total RNA per reaction, following protocol 4304965 of Applied Biosystems (Foster City, Calif.).
Primers for expression analysis using TaqMan® assay (Applied Biosystems, Foster City, Calif.) were prepared according to the TaqMan® protocols, and the following criteria: a) primer pairs were designed to span introns to eliminate genomic contamination, and b) each primer pair produced only one product. Expression analysis was performed using a 7900HT instrument.
TaqMan® reactions were carried out following manufacturer's protocols, in 25 μl total volume for 96-well plates and 10 μl total volume for 384-well plates, using 300 nM primer and 250 nM probe, and approximately 25 ng of cDNA. The standard curve for result analysis was prepared using a universal pool of human cDNA samples, which is a mixture of cDNAs from a wide variety of tissues so that the chance that a target will be present in appreciable amounts is good. The raw data were normalized using 18S rRNA (universally expressed in all tissues and cells).
For each expression analysis, tumor tissue samples were compared with matched normal tissues from the same patient. A gene was considered overexpressed in a tumor when the level of expression of the gene was 2 fold or higher in the tumor compared with its matched normal sample. In cases where normal tissue was not available, a universal pool of cDNA samples was used instead. In these cases, a gene was considered overexpressed in a tumor sample when the difference of expression levels between a tumor sample and the average of all normal samples from the same tissue type was greater than 2 times the standard deviation of all normal samples (i.e., Tumor−average (all normal samples)>2×STDEV (all normal samples)).
Results are shown in Table 2. Number of pairs of tumor samples and matched normal tissue from the same patient are shown for each tumor type. Percentage of the samples with at least two-fold overexpression for each tumor type is provided. A modulator identified by an assay described herein can be further validated for therapeutic effect by administration to a tumor in which the gene is overexpressed. A decrease in tumor growth confirms therapeutic utility of the modulator. Prior to treating a patient with the modulator, the likelihood that the patient will respond to treatment can be diagnosed by obtaining a tumor sample from the patient, and assaying for expression of the gene targeted by the modulator. The expression data for the gene(s) can also be used as a diagnostic marker for disease progression. The assay can be performed by expression analysis as described above, by antibody directed to the gene target, or by any other available detection method.
KIF23 was highly expressed (P<0.001) in breast tumors, breast basal tumors, breast luminal tumors, colon AC tumors, head/neck tumors, liver tumors, lung tumors, lung LCLC tumors, lung SCC tumors, ovary tumors, pancreas tumors, skin tumors, stomach tumors, and uterine tumors. KIF23 was overexpressed (0.05>P>0.001) in colon tumors, lung AC3 tumors, lung SCLC tumors and lymphomas. KIF23 was underexpressed (0.05>P>0.001) in kidney tumors.
RNAi experiments were carried out to knock down expression of various KIF23 sequences in various cell lines using small interfering RNAs (siRNA, Elbashir et al, supra). The following cell lines were used in the experiments: A549 lung cancer cells, MBA-MB231T breast carcinoma cells, HCT116 colorectal cancer cells, A2780 human ovarian carcinoma cells and HELA cervical cancer cells.
Effect of KIF23 RNAi on cell proliferation and growth. BrdU, Caspase 3, and Cell Titer-Glo™ assays, as described above, were employed to study the effects of decreased KIF23 expression on cell proliferation.
Results: RNAi of KIF23 decreased cell proliferation in all cell lines tested.
Standard colony growth assays, as described above, were employed to study the effects of decreased KIF23 expression on cell growth.
Results: RNAi of KIF23 decreased proliferation in all cell lines tested.
Effect of KIF23 RNAi on Apoptosis.
Multiple parameter apoptosis assay, as described above, was also used to study the effects of decreased KIF23 expression on apoptosis, using Caspase 3 cleavage readout.
Results of this experiment indicated that RNAi of KIF23 increased apoptosis in all cell lines tested.
Transcriptional reporter assays. Effects of overexpressed KIF23 on expression of various transcription factors was also studied. In this assay, rat intestinal epithelial cells (RIEs) or NIH3T3 cells were co-transfected with reporter constructs containing various transcription factors and luciferase along with KIF23. Luciferase intensity was then measured as the readout for transcriptional activation due to overexpression of the KIF23. Overexpressed KIF23 activated AP1, NFKB, and SRE (Serum Response element).
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/US06/43865 | 11/9/2006 | WO | 00 | 9/10/2008 |
| Number | Date | Country | |
|---|---|---|---|
| 60735612 | Nov 2005 | US |
