WO2005095977A1 - Agents de diagnostic et therapeutiques pour des maladies associees a proteine kinase p21/cdc42/rac1 - Google Patents

Agents de diagnostic et therapeutiques pour des maladies associees a proteine kinase p21/cdc42/rac1 Download PDF

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Publication number
WO2005095977A1
WO2005095977A1 PCT/EP2005/001749 EP2005001749W WO2005095977A1 WO 2005095977 A1 WO2005095977 A1 WO 2005095977A1 EP 2005001749 W EP2005001749 W EP 2005001749W WO 2005095977 A1 WO2005095977 A1 WO 2005095977A1
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diseases
pakl
polypeptide
activity
muscle
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PCT/EP2005/001749
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Stefan Golz
Ulf Brüggemeier
Andreas Geerts
Holger Summer
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Bayer Healthcare Ag
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/91—Transferases (2.)
    • G01N2333/912—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • G01N2333/91205—Phosphotransferases in general
    • G01N2333/9121—Phosphotransferases in general with an alcohol group as acceptor (2.7.1), e.g. general tyrosine, serine or threonine kinases
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value

Definitions

  • the present invention is in the field of molecular biology, more particularly, the present invention relates to nucleic acid sequences and amino acid sequences of a human PAKl and its regulation for the treatment of cardiovascular diseases, dermatological diseaes, endocrinological diseases, metabolic diseases, inflammation, gastroenterological diseases, cancer, hematological diseases, respiratory diseases, muscle-skeleton diseases, neurological diseases and urological diseases in mammals.
  • PAKl is a member of the enzyme group of kinases [Brown et al. (1996), Knaus et al. (1995), Sanders et al. (1999), Parrini et al. (2002), Vadlamudi et al. (2002), Lei et al. (2000), Li et al. (2003), WO0281638, O0136602, WO0228999, WO0060062, WO9953036].
  • Kinases are enzymes, which catalyse the transfer of a phosphate group (phosphorylation) from a donor (mainly ATP) onto an acceptor molecule's nucleophilic functional group, such as hydroxy-, carboxy-, guanidino-, thiol-, or imidazole- groups.
  • a phosphate group phosphorylation
  • acceptor molecule's nucleophilic functional group such as hydroxy-, carboxy-, guanidino-, thiol-, or imidazole- groups.
  • Kinases can act on on a variety of molecules like small metabolites, or proteins (protein kinases).
  • kinases that phosphorylate small organic molecules often play important roles in metabolic pathways such as glycolosis (e.g. hexokinase, glucokinase, or phosphofructokinase), or in anabolic pathways.
  • glycolosis e.g. hexokinase, glucokinase, or phosphofructokinase
  • Protein kinases play critical roles in cellular signal transduction.
  • Cellular signal transduction is a fundamental mechanism whereby extracellular stimuli are relayed to the interior of cells and subsequently regulate diverse cellular processes.
  • One of the key biochemical mechanisms of signal transduction involves the reversible phosphorylation of proteins. Phosphorylation of polypeptides regulates tfie activity of mature proteins by altering their structure and function. Phosphate most often resides on the hydroxyl moiety (--OH) of serine, threonine, or tyrosine amino acids in proteins.
  • kinases regulate many different cell proliferation, differentiation, and signaling processes by adding phosphate groups to proteins. Uncontrolled signaling has been implicated in a variety of disease conditions including inflammation, cancer, arteriosclerosis, and psoriasis. Reversible protein phosphorylation is the main strategy for controlling activities of eukaryotic cells.
  • the high energy phosphate which drives activation, is generally transferred from adenosine triphosphate molecules (ATP) to a particular protein by protein kiriases and removed from that protein by protein phosphatases.
  • ATP adenosine triphosphate molecules
  • Phosphorylation occurs in response to extracellular signals (hormones, neurotransmitters, growth and differentiation factors, etc), cell cycle checkpoints, and environ- mental or nutritional stresses and is roughly analogous to turning on a molecular switch.
  • the appropriate protein kinase activates a metabolic enzyme, regulatory protein, receptor, cytoskeletal protein, ion channel or pump, or transcription factor.
  • the kinases comprise the largest known protein group, a superfamily of enzymes with widely varied functions and specificities. They are usually named after their substrate, their regulatory molecules, or some aspect of a mutant phenotype. With- regard to substrates, the protein kinases may be roughly divided into two groups; those that phosphorylate tyrosine residues (protein tyrosine kinases, PTK) and those that phosphorylate seri-ne or threonine residues (serine/threonine kinases, STK). A few protein kinases have dual specificity and phosphorylate threonine and tyrosine residues. Almost all kinases contain a similar 2-50-300 amino acid catalytic domain.
  • the N-terminal domain which contains subdomains I-IV, generally folds into a two-lobed structure, which binds and orients the ATP (or GTP) donor molecule.
  • the larger C terminal lobe which contains subdomains VI-XI, binds the protein substrate and carries out the transfer of the gamma phosphate from ATP to the hydroxyl group of a serine, -hreonine, or tyrosine residue.
  • Subdomain V spans the two lobes.
  • the kinases may be categorized into families by the different amino acid sequences (generally between 5 and 100 residues) located on either side of, or inserted into loops of, the kinase domain. These added amino acid sequences allow the regulation of each kinase as it recognizes and interacts with its target protein.
  • the primary structure of the kinase domains is conserved and can be further subdivided into 11 subdomains. Each of the 11 subdomains contains specific residues and motifs or patterns of amino acids that are characteristic of that subdomain and are highly conserved [Hardie, G. and Hanks, S. (1995)].
  • the second messenger dependent protein kinases primarily mediate the effects of second messengers such as cyclic AMP (cAMP), cyclic GMP, inositol triphosphate, phosphatidylinositol, 3,4,5-triphosphate, cyclic-ADPribose, arachidonic acid, diacylglycerol and calcium-calmodulin.
  • cyclic AMP cyclic AMP
  • GMP cyclic GMP
  • inositol triphosphate phosphatidylinositol
  • cyclic-ADPribose cyclic-ADPribose
  • arachidonic acid diacylglycerol
  • calcium-calmodulin calcium-calmodulin.
  • the cyclic-AMP dependent protein kinases (PKA) are important members of the STK family. Cyclic-AMP is an intracellular mediator of hormone action in all prokaryotic and animal cells that have been studied.
  • Such hormone-induced cellular responses include thyroid hormone secretion, cortisol secretion, progesterone secretion, glycogen brea -down, bone resorption, and regulation of heart rate and force of heart muscle contraction.
  • PKA is found in all animal cells and is thought to account for the effects of cyclic-AMP in most of these cells. Altered PKA expression is implicated in a variety of disorders and diseases including cancer, thyroid disorders, diabetes, atherosclerosis, and cardiovascular disease [Isselbacher, K. J. et al. (1994)].
  • CaM dependent protein kinases are also members of STK family. Calmodulin is a calcium receptor that mediates many calcium regulated processes by binding to target proteins in response to the binding of calcium. The principle target protein in these processes is CaM dependent protein kinases. CaM-kinases are involved in regulation of smooth muscle contraction (MLC kinase), glycogen breakdown (phosphorylase kinase), and neuro- transmission (CaM kinase I and CaM kinase II).
  • MLC kinase smooth muscle contraction
  • phosphorylase kinase glycogen breakdown
  • CaM kinase II neuro- transmission
  • CaM kinase I phosphorylates a variety of substrates including the neurotransmitter related proteins synapsin I and ⁇ , the gene transcription regulator, CREB, and the cystic fibrosis conductance regulator protein, CFTR [Haribabu, B. et al. (1995)].
  • CaM II kinase also phosphorylates synapsin at different sites, and controls the synthesis of catecholamines in the brain through phosphorylation and activation of tyrosine hydroxylase. Many of the CaM kinases are activated by phosphorylation in addition to binding to CaM. The kinase may autophosphorylate itself, or be phosphorylated by another kinase as part of a "kinase cascade".
  • AMPK 5'-AMP-activated protein kinase
  • Mammalian AMPK is a regulator of fatty acid and sterol synthesis through phosphorylation of the enzymes acetyl-CoA carboxylase and hydroxymethylglutaryl-CoA reductase and mediates responses of these pathways to cellular stresses such as heat shock and depletion of glucose and ATP.
  • AMPK is a heterotrimeric complex comprised of a catalytic alpha subunit and two non-catalytic beta and gamma subunits that are believed to regulate the activity of the alpha subunit.
  • Subunits of AMPK have a much wider distribution in non-lipogenic tissues such as brain, heart, spleen, and lung than expected. This distribution suggests that its role may extend beyond regulation of lipid metabolism alone.
  • MAP kinases are also members of the STK family. MAP kinases also regulate intracellular signaling pathways. They mediate signal transduction from the cell surface to the nucleus via phosphorylation cascades. Several subgroups have been identified, and each manifests different substrate specificities and responds to distinct extracellular stimuli [Egan, S. E. and Weinberg, R. A. (1993)]. MAP kinase signaling pathways are present in mammalian cells as well as in yeast.
  • the extracellular stimuli that activate mammalian pathways include epidermal growth factor (EGF), ultraviolet light, hyperosmolar medium, heat shock, endotoxic lipopoly- saccharide (LPS), and pro-inflammatory cytokines such as tumor necrosis factor (TNF) and interleukin-1 (IL-1).
  • EGF epidermal growth factor
  • LPS endotoxic lipopoly- saccharide
  • cytokines such as tumor necrosis factor (TNF) and interleukin-1 (IL-1).
  • PRK proliferation-related kinase
  • PRK is a serum/cytokine inducible STKL that is involved in regulation of the cell cycle and cell proliferation in human megakaroytic cells [Li, B. et al. (1996)].
  • PRK is related to the polo (derived from humans polo gene) family of STKLs implicated in cell division.
  • PRK is downregulated in lung tumor tissue and may be a proto-oncogene whose deregulated expression in normal tissue leads to oncogenic transformation.
  • Altered MAP kinase expression is implicated in a variety of disease conditions including cancer, inflammation, immune disorders, and disorders affecting growth and development.
  • CDKs The cyclin-dependent protein kinases
  • CycHns are small regulatory proteins that act by binding to and activating CDKs that then trigger various phases of the cell cycle by phosphorylating and activating selected proteins involved in the mitotic process.
  • CDKs are unique in that they require multiple inputs to become activated. In addition to the binding of cyclin, CDK activation requires the phosphorylation of a specific threonine residue and the dephosphorylation of a specific tyrosine residue.
  • Protein tyrosine kinases specifically phosphorylate tyrosine residues on their target proteins and may be divided into transmembrane, receptor PTKs and nontransmembrane, non- receptor PTKs.
  • Transmembrane protein-tyrosine kinases are receptors for most growth factors. Binding of growth factor to the receptor activates the transfer of a p-hosphate group from ATP to selected tyrosine side chains of the receptor and other specific proteins.
  • Growth factors (GF) associated with receptor PTKs include; epidermal GF, platelet-derived GF, fibroblast GF, hepatocyte GF, insulin and insulin-like GFs, nerve GF, vascular endothelial GF, and macrophage colony stimulating factor.
  • Non-receptor PTKs lack transmembrane regions and, instead, form complexes with the intracellular regions of cell surface receptors.
  • Such receptors that fmnction through non-receptor PTKs include those for cytokines, hormones (growth hormone and prolactin) and antigen-specific receptors on T and B lymphocytes.
  • TaqMan is a recently developed technique, in which the release of a fluorescent reporter dye from a hybridisation probe in real-time during a polymerase chain reaction (PCR) is propo-rtional to the accumulation of the PCR product. Quantification is based on the early, linear part of the reaction, and by determining the threshold cycle (CT), at which fluorescence above background is first detected.
  • CT threshold cycle
  • Gene expression technologies may be useful in several areas of drug discovery and clevelopment, such as target identification, lead optimization, and identification of mechanisms of action.
  • the TaqMan technology can be used to compare differences between expression profiles of normal tissue and diseased tissue.
  • Expression profiling has been used in identifying genes, v ⁇ hich are up- or downregulated in a variety of diseases.
  • An interesting application of expressio t profiling is temporal monitoring of changes in gene expression during disease progression and drxig treatment or in patients versus healthy individuals.
  • the premise in this approach is that changes in pattern of gene expression in response to physiological or environmental stimuli (e.g., drugs) may serve as indirect clues about disease-causing genes or drug targets.
  • physiological or environmental stimuli e.g., drugs
  • the effects of drugs with established efficacy on global gene expression patterns may provide a guidepost, or a genetic signature, against which a new drug candidate can be compared.
  • the nucleotide sequence of PAKl is accessible in public databases by the accession number NM_002576 and is given in SEQ ID NO: 1.
  • the amino acid sequence of PAKl is depicted in SEQ ID NO: 2.
  • PAK proteins are critical effectors that link RhoGTPases to cytoskeleton reorganization and nuclear signaling.
  • PAK proteins a family of serine/threonine p21 -activating kinases, include PAKl, PAK2, PAK3 and PAK4. These proteins serve as targets for the small G-TP binding proteins Cdc42 and Rac and have been implicated in a wide range of biological activities.
  • PAKl regulates cell motility and morphology.
  • PAKl is a member of a family of serine/threonine kinases related to yeast Ste20. Brown et al. (1996) used PCR based on the sequence of rat PAKl to clone a human PAKl cIXNA from a placental cDNA library. The human PAKl gene encodes a 545-amino acid polypeptide that is 98% identical to rat PAKl and 52% identical to yeast Ste20. Brown et al. (1996) found that human PAKl can replace Ste20 in the mating response pathway of yeast. Activity of PAKl is induced by coexpression with RAC1 or CDC42. PAK is believed to act directly on the JNK1 MAP kinase pathway.
  • PAKl protein promotes the disassembly of stress fibers and focal adhesions.
  • Sanders et al. (1999) demonstrated that, in baby hamster kidney-21 and HeLa cells expressing constitutively active PAKl, MLCK activity and myosin light-chain phosphorylation were decreased, and cell spreading was inhibited. These results indicated that MLCK is a target for PAKl, and that PAKs may regulate cytoskeletal dynamics by decreasing MLCK activity and myosin light-chain phosphorylation.
  • FLNA filamin A
  • G beta gamma binds PAKl and, via PAK-associated PIX alpha, activates Cdc42 which, in turn, activates PAKl.
  • PAKl is not only an effector for Cdc42, but it also functions as a scaffold protein required for Cdc42 activation [Li et al. (2003)]
  • PAKl copy number gains were observed in 30% of ovarian carcinomas and PAKl protein was expressed in 85% of tumors. PAKl gains were associated with high grade [Schraml et al. (2003)].
  • PAKl is published in WO0281638, WO0136602, WO0228999, WO0060062 and WO9953036.
  • PAKl shows the highest homology (80%) to the human Serine/threonine-protein kinase PAK 3 as shown in example 1.
  • the invention relates to novel disease associations of PAKl polypeptides and polynucleotides.
  • the invention also relates to novel methods of screening for therapeutic agents for the treatment of cardiovascular diseases, dermatological diseaes, endocrinological diseases, metabolic diseases, inflammation, gastroenterological diseases, cancer, hematological diseases, respiratory diseases, muscle-skeleton diseases, neurological diseases and urological diseases in a mammal.
  • the invention also relates to pharmaceutical compositions for the treatment of cardiovascular diseases, dermatological diseaes, endocrinological diseases, metabolic diseases, inflammation, gastroenterological diseases, cancer, hematological diseases, respiratory diseases, muscle-skeleton diseases, neurological diseases and urological diseases in a mammal comprising a PAKl polypeptide, a PAKl polynucleotide, or regulators of PAKl or modulators of PAKl activity.
  • the invention further comprises methods of diagnosing cardiovascular diseases, dermatological diseaes, endocrinological diseases, metabolic diseases, inflammation, gastroenterological diseases, cancer, hematological diseases, respiratory diseases, muscle-skeleton diseases, neurological diseases and urological diseases in a mammal.
  • Fig. 1 shows the nucleotide sequence of a PAKl polynucleotide (SEQ ID NO: 1).
  • Fig. 2 shows the amino acid sequence of a PAKl polypeptide (SEQ ID NO: 2).
  • Fig. 3 shows the nucleotide sequence of a primer useful for the invention (SEQ ID NO: 3).
  • Fig. 4 shows the nucleotide sequence of a primer useful for the invention (SEQ ID NO: 4).
  • Fig. 5 shows a nucleotide sequence useful as a probe to detect proteins of the invention (SEQ ID NO: 5).
  • oligonucleotide is a stretch of nucleotide residues which has a sufficient number of bases to be used as an oligomer, amplimer or probe in a polymerase chain reaction (PCR). Oligonucleotides are prepared from genomic or cDNA sequence and are used to amplify, reveal, or confirm the presence of a similar DNA or RNA in a particular cell or tissue. Oligonucleotides or oligomers comprise portions of a DNA sequence having at least about 10 nucleotides and as many as about 35 nucleotides, preferably about 25 nucleotides.
  • Probes may be derived from naturally occurring or recombinant single- or double-stranded nucleic acids or may be chemically synthesized. They are useful in detecting the presence of identical or similar sequences. Such probes may be labeled with reporter molecules using nick translation, Klenow fill-in reaction, PCR or other methods well known in the art. Nucleic acid probes may be used in southern, northern or in situ hybridizations to determine whether DNA or RNA encoding a certain protein is present in a cell type, tissue, or organ.
  • a “fragment of a polynucleotide” is a nucleic acid that comprises all or any part of a given nucleotide molecule, the fragment having fewer nucleotides than about 6 kb, preferably fewer than about 1 kb.
  • Reporter molecules are radionuclides, enzymes, fluorescent, chemiluminescent, or chromogenic agents which associate with a particular nucleotide or amino acid sequence, thereby establishing the presence of a certain sequence, or allowing for the quantification of a certain sequence.
  • Chimeric molecules may be constructed by introducing all or part of the nucleotide sequence of this invention into a vector containing additional nucleic acid sequence which might be expected to change any one or several of the followmg PAKl characteristics: cellular location, distribution, ligand-binding affinities, interchain affinities, degradation/turnover rate, signaling, etc.
  • PAKl polypeptide refers to those forms, fragments, or domains of a PAKl polypeptide which retain the biological and/or antigenic activity of a PAKl polypeptide.
  • “Naturally occurring PAKl polypeptide” refers to a polypeptide produced by cells which have not been genetically engineered and specifically contemplates various polypeptides arising from post- translational modifications of the polypeptide including but not limited to acetylation, carboxylation, glycosylation, phosphorylation, lipidation and acylation.
  • Derivative refers to polypeptides which have been chemically modified by techniques such as ubiquitination, labeling (see above), pegylation (derivatization with polyethylene glycol), and chemical insertion or substitution of amino acids such as ornithine which do not normally occur in human proteins.
  • Constant amino acid substitutions result from replacing one amino acid with another having similar structural and/or chemical properties, such as the replacement of a leucine with an isoleucine or valine, an aspartate with a glutamate, or a threonine with a serine.
  • “Insertions” or “deletions” are typically in the range of about 1 to 5 amino acids. The variation allowed may be experimentally determined by producing the peptide synthetically while systematically making insertions, deletions, or substitutions of nucleotides in the sequence using recombinant DNA techniques.
  • a “signal sequence” or “leader sequence” can be used, when desired, to direct the polypeptide through a membrane of a cell.
  • Such a sequence may be naturally present on the polypeptides of the present invention or provided from heterologous sources by recombinant DNA techniques.
  • Oligopeptide is a short stretch of amino acid residues and may be expressed from an oligonucleotide. Oligopeptides comprise a stretch of amino acid residues of at least 3, 5, 10 amino acids and at most 10, 15, 25 amino acids, typically of at least 9 to 13 amino acids, and of sufficient length to display biological and/or antigenic activity.
  • inhibitor is any substance which retards or prevents a chemical or physiological reaction or response. Common inhibitors include but are not limited to antisense molecules, antibodies, and antagonists.
  • Standard expression is a quantitative or qualitative measurement for comparison. It is based on a statistically appropriate number of normal samples and is created to use as a basis of comparison when performing diagnostic assays, running clinical trials, or following patient treatment profiles.
  • Animal as used herein may be defined to include human, domestic (e.g., cats, dogs, etc.), agricultural (e.g., cows, horses, sheep, etc.) or test species (e.g., mouse, rat, rabbit, etc.).
  • a "PAKl polynucleotide”, within the meaning of the invention, shall be understood as being a nucleic acid molecule selected from a group consisting of
  • nucleic acid molecules encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 2,
  • nucleic acid molecules comprising the sequence of SEQ ID NO: 1,
  • nucleic acid molecules the complementary strand of which hybridizes under stringent conditions to a nucleic acid molecule of (i), (ii), or (iii);
  • nucleic acid molecules the sequence of which differs from the sequence of a nucleic acid molecule of (iii) due to the degeneracy of the genetic code;
  • polypeptide encoded by said nucleic acid molecule has PAKl activity.
  • PAKl polypeptide within the meaning of the invention, shall be understood as being a polypeptide selected from a group consisting of
  • polypeptides which show at least 99%, 98%, 95%, 90%, or 80% homology with a polypeptide of (i), (ii), or (iii);
  • polypeptide has PAKl activity.
  • nucleotide sequences encoding a PAKl have numerous applications in techniques known to those skilled in the art of molecular biology. These techniques include use as hybridization probes, use in the construction of oligomers for PCR, use for chromosome and gene mapping, use in the recombinant production of PAKl, and use in generation of antisense DNA or RNA, their chemical analogs and the like. Uses of nucleotides encoding a PAKl disclosed herein are exemplary of known techniques and are not intended to limit their use in any technique known to a person of ordinary skill in the art.
  • nucleotide sequences disclosed herein may be used in molecular biology techniques that have not yet been developed, provided the new techniques rely on properties of nucleotide sequences that are currently known, e.g., the triplet genetic code, specific base pair interactions, etc.
  • PAKl - encoding nucleotide sequences may be produced. Some of these will only bear minimal homology to the nucleotide sequence of the known and naturally occurring PAKl.
  • the invention has specifically contemplated each and every possible variation of nucleotide sequence that could be made by selecting combinations based on possible codon choices. These combinations are made in " accordance with the standard triplet genetic code as applied to the nucleotide sequence of naturally occurring PAKl, and all such variations are to be considered as being specifically disclosed.
  • nucleotide sequences which enc'ode a PAKl, its derivatives or its variants are preferably capable of hybridizing to the nucleotide sequence of the naturally occurring PAKl polynucleotide under stringent conditions, it may be advantageous to produce nucleotide sequences encoding PAKl polypeptides or its derivatives possessing a substantially different codon usage. Codons can be selected to increase the rate at which expression of the peptide occurs in a particular prokaryotic or eukaryotic expression host in accordance with the frequency with which particular codons are utilized by the host.
  • RNA transcripts having more desirable properties such as a greater half-life, than transcripts produced from the naturally occurring sequence.
  • Nucleotide sequences encoding a PAKl polypeptide may be joined to a variety of other nucleotide sequences by means of well established recombinant DNA techniques.
  • Useful nucleotide sequences for joining to PAKl polynucleotides include an assortment of cloning vectors such as plasmids, cosmids, lambda phage derivatives, phagemids, and the like.
  • Vectors of interest include expression vectors, replication vectors, probe generation vectors, sequencing vectors, etc.
  • vectors of interest may contain an origin of replication functional in at least one organism, convenient restriction endonuclease sensitive sites, and selectable markers for one or more host cell systems.
  • Another aspect of the subject invention is to provide for PAKl -specific hybridization probes capable of hybridizing with naturally occurring nucleotide sequences encoding PAKl. Such probes may also be used for the detection of similar kinase encoding sequences and should preferably show at least 40% nucleotide identity to PAKl polynucleotides.
  • the hybridization probes of the subject invention may be derived from the nucleotide sequence presented as SEQ ID NO: 1 or from genomic sequences including promoter, enhancers or introns of the native gene. Hybridization probes may be labelled by a variety of reporter molecules using techniques well known in the art.
  • the invention relates to nucleic acid sequences that hybridize with such PAKl encoding nucleic acid sequences under stringent conditions.
  • Stringent conditions refers to conditions that allow for the hybridization of substantially related nucleic acid sequences. For instance, such conditions will generally allow hybridization of sequence with at least about 85% sequence identity, preferably with at least about 90% sequence identity, more preferably with at least about 95% sequence identity. Hybridization conditions and probes can be adjusted in well-characterized ways to achieve selective hybridization of human- derived probes. Stringent conditions, within the meaning of the invention are 65°C in a buffer containing 1 mM EDTA, 0.5 M NaHP0 4 (pH 7.2); 7 % (w/v) SDS.
  • Nucleic acid molecules that will hybridize to PAKl polynucleotides under stringent conditions can be identified functionally.
  • examples of the uses for hybridization probes include: histochemical uses such as identifying tissues that express PAKl; measuring mRNA levels, for instance to identify a sample's tissue type or to identify cells that express abnormal levels of PAKl; and detecting polymorphisms of PAKl.
  • PCR provides additional uses for oligonucleotides based upon the nucleotide sequence which encodes PAKl.
  • probes used in PCR may be of recombinant origin, chemically synthesized, or a mixture of both.
  • Oligomers may comprise discrete nucleotide sequences employed under optimized conditions for identification of PAKl in specific tissues or diagnostic use. The same two oligomers, a nested set of oligomers, or even a degenerate pool of oligomers may be employed under less stringent conditions for identification of closely related DNAs or RNAs.
  • PCR primers i.e., preparations of primers that are heterogeneous at given sequence locations, can be designed to amplify nucleic acid sequences that are highly homologous to, but not identical with PAKl.
  • Strategies are now available that allow for only one of the primers to be required to specifically hybridize with a known sequence.
  • appropriate nucleic acid primers can be ligated to the nucleic acid sought to be amplified to provide the hybridization partner for one of the primers. In this way, only one of the primers need be based on the sequence of the nucleic acid sought to be amplified.
  • PCR methods for amplifying nucleic acid will utilize at least two primers.
  • One of these primers will be capable of hybridizing to a first strand of the nucleic acid to be amplified and of priming enzyme-driven nucleic acid synthesis in a first .direction.
  • the other will be capable of hybridizing the reciprocal sequence of the first strand (if tne sequence to be amplified is single stranded, this sequence will initially be hypothetical, but will be synthesized in the first amplification cycle) and of priming nucleic acid synthesis from that strand iri the direction opposite the first direction and towards the site of hybridization for the first primer.
  • Conditions for conducting such amplifications particularly under preferred stringent hybridization conditions, are well known.
  • RNA polymerase as T7 or SP6 RNA polymerase and the appropriate reporter molecules.
  • nucleic acid sequence can be inserted into any of the many available DNA vectors and their respective host cells using techniques which are well known in the art.
  • synthetic chemistry may be used to introduce mutations into the nucleotide sequence. Alternately, a portion of sequence in which a mutation is desired can be synthesized and recombined with longer portion of an existing genomic or recombinant sequence.
  • PAKl polynucleotides may be used to produce a purified oligo-or polypeptide using well known methods of recombinant DNA technology.
  • the oligopeptide may be expressed in a variety of host cells, either prokaryotic or eukaryotic. Host cells may be from the same species from which the nucleotide sequence was derived or from a different species. Advantages of producing an oligonucleotide by recombinant DNA technology include obtaining adequate amounts of the protein for purification and the availability of simplified purification procedures.
  • Chromosome-based techniques such as comparative genomic hybridization (CGH) and fluorescent in situ hybridization (FISH) facilitate efforts to cytogenetically localize genomic regions that are altered in tumor cells. Regions of genomic alteration can be nanowed further using loss of heterozygosity analysis (LOH), in which. disease DNA is analyzed and compared with normal DNA for the loss of a heterozygous polymorphic marker.
  • LH loss of heterozygosity analysis
  • RFLPs restriction fragment length polymo ⁇ hisms
  • Johnson, (1989) or hypervariable minisatellite DNA [Barnes, 2000].
  • a gene sequence contained in all samples at relatively constant quantity is typically utilized for sample amplification efficiency normalization.
  • This approach suffers from several drawbacks.
  • the method requires that each sample has equal input amounts of the nucleic acid and that the amplification efficiency between samples is identical until the time of analysis.
  • QC-PCR quantitative competitive PCR
  • An internal control competitor in each reaction [Piatak, (1993), BioTechniques].
  • the efficiency of each reaction is normalized to the internal competitor.
  • a known amount of internal competitor is typically added to each sample.
  • the unknown target PCR product is compared with the known competitor PCR product to obtain relative. quantitation.
  • a difficulty with this general approach lies in developing an internal control that amplifies with the same efficiency than the target molecule.
  • Fluorogenic nuclease assays are a real time quantitation method that uses a probe to monitor formation of amplification product.
  • the basis for this method of monitoring the formation of amplification product is to measure continuously PCR product accumulation using a dual-labelled fluorogenic oligonucleotide probe, an approach frequently refereed to in the literature simply as the "TaqMan method” [Piatak, (1993), Science; Heid, (1996); Gibson, (1996); Holland. (1991)].
  • the probe used in such assays is typically a short (about 20-25 bases) oligonucleotide that is labeled with two different fluorescent dyes.
  • the 5' terminus of the probe is attached to a reporter dye and the 3' terminus is attached to a quenching dye, although the dyes could be attached at other locations on the probe as well.
  • the probe is designed to have at least substantial sequence complementarity with the probe binding site. Upstream and downstream PCR primers which bind to flanking regions of the locus are added to the reaction mixture. When the probe is intact, energy transfer between the two fluorophors occurs and the quencher quenches emission from the reporter.
  • the probe is cleaved by the 5' nuclease activity of a nucleic acid polymerase such as Taq polymerase, thereby releasing the reporter from the oligonucleotide-quencher and resulting in an increase of reporter emission intensity which can be measured by an appropriate detector.
  • a nucleic acid polymerase such as Taq polymerase
  • One detector which is specifically adapted for measuring fluorescence emissions such as those created during a fluorogenic assay is the ABI 7700 or 4700 HT manufactured by Applied Biosystems, Inc. in Foster City, Calif.
  • the ABI 7700 uses fiber optics connected with each well in a 96-or 384 well PCR tube anangement.
  • the instrument includes a laser for exciting the labels and is capable of measuring the fluorescence spectra intensity from each tube with continuous monitoring during PCR amplification. Each tube is re-examined every 8.5 seconds.
  • Computer software provided with the instrument is capable of recording the fluorescence intensity of reporter and quencher over the course of the amplification. The recorded values will then be used to calculate the increase in normalized reporter emission intensity on a continuous basis. The increase in emission intensity is plotted versus time, i.e., the number of amplification cycles, to produce a continuous measure of amplification.
  • the amplification plot is examined at a point during the log phase of product accumulation. This is accomplished by assigning a fluorescence threshold intensity above background and determining the point at which each amplification plot crosses the threshold (defined as the threshold cycle number or Ct). Differences in threshold cycle number are used to quantify the relative amount of PCR target contained within each tube. Assuming that each reaction functions at 100% PCR efficiency, a difference of one Ct represents a two-fold difference in the amount of starting template.
  • the fluorescence value can be ' used in conjunction with a standard curve to determine the amount of amplification product present.
  • amplification product which is double stranded
  • amplification product binds dye molecules in solution to form a complex.
  • dyes it is possible to distinguish between dye molecules free in solution and dye molecules bound to amplification product.
  • certain dyes fluoresce only when bound to amplification product.
  • dyes which can be used in methods of this general type include, but are not limited to, Syber Green.TM. and Pico Green from Molecular Probes, Inc. of Eugene, Oreg.
  • These detection methods involve some alteration to the structure or conformation of a probe hybridized to the locus between the amplification primer pair.
  • the alteration is caused by the template-dependent extension catalyzed by a nucleic acid polymerase during the amplification process.
  • the alteration generates a detectable signal which is an indirect measure of the amount of amplification product formed.
  • some methods involve the degradation or digestion of the probe during the extension reaction. These methods are a consequence of the 5 '-3' nuclease activity associated with some nucleic acid polymerases. Polymerases having this activity cleave mononucleotides or small oligonucleotides from an oligonucleotide probe annealed to its complementary sequence located within the locus.
  • the 3' end of the upstream primer provides the initial binding site for the nucleic acid polymerase.
  • the nucleic acid polymerase displaces a portion of the '5' end of the probe and through its nuclease activity cleaves mononucleotides or oligonucleotides from the probe.
  • the upstream primer and the probe can be designed such that they anneal to the complementary strand in close proximity to one another. In fact, the 3' end of the upstream primer and the 5' end of the probe may abut one another. In this situation, extension of the upstream primer is not necessary in order for the nucleic acid polymerase to begin cleaving the probe. Li the case in which intervening nucleotides separate the upstream primer and the probe, extension of the primer is necessary before the nucleic acid polymerase encounters the 5' end of the probe.
  • the 5 '-3' exonuclease activity of the nucleic acid polymerase begins cleaving mononucleotides or oligonucleotides from the 5' end of the probe. Digestion of the probe continues until the remaining portion of the probe dissociates from the complementary strand.
  • the two end sections can hybridize with each other to form a hairpin loop.
  • the reporter and quencher dye are in sufficiently close proximity that fluorescence from the reporter dye is effectively quenched b the quencher dye.
  • Hybridized probe in contrast, results in a linearized conformation in which the extent of quenching is decreased.
  • the labeled probe is selected so that its sequence is substantially complementary to a segment of the test locus or a reference locus. As indicated above, the nucleic acid site to which the probe binds should be located between the primer binding sites for the upstream and downstream amplification primers.
  • the primers used in the amplification are selected so as to be capable of hybridizing to sequences at flanking regions of the locus being amplified.
  • the primers are chosen to have at least substantial complementarity with the different strands of the nucleic acid being amplified.
  • the primers are selected in such that they flank the probe, i.e. are located upstream and. downstream of the probe.
  • the primer must have sufficient length so that it is capable of priming the synthesis of extension products in the presence of an agent for polymerization.
  • the length and composition of the primer depends on many parameters, including, for example, the temperature at which the annealing reaction is conducted, proximity of the probe binding site to that of the primer, relative concentrations of the primer and probe and the particular nucleic acid composition of the probe.
  • the primer typically includes 15-30 nucleotides.
  • the length of the primer may be more or less depending on the complexity of the primer binding site and the factors listed above.
  • the labels used for labeling the probes or primers of the cunent invention and which can provide the signal conesponding to the quantity of amplification product can take a variety of forms.
  • a fluorescent signal is one signal which can be measured. However, measurements may also be made, for example, by . .
  • labels which can be employed include, but are not limited to, fluorophors, chromophores, radioactive isotopes, electron dense reagents, enzymes, and ligands having specific binding partners (e.g., biotin-avidiii).
  • Labels may be attached to the probe or primer using a variety of techniques and can be attached at the 5' end, and/or the 3' end and/or at an internal. nucleotide.
  • the label can also be attached to spacer arms of various sizes which are attached to the probe or primer. These spacer arms are useful for obtaining a desired distance between multiple labels attached to the probe or primer.
  • a single label may be utilized; whereas, in other instances, such as with the 5' fluorogenic nuclease assays for example, two or more labels are attached to the probe.
  • the probe includes multiple labels, it is generally advisable to maintain spacing between the labels which is sufficient to permit separation of the labels during digestion of the probe through the 5 -3' nuclease activity of the nucleic acid polymerase.
  • a number of diseases are associated with, changes in the copy number of a certain gene.
  • the real-time PCR method can be used to determine if the patient has copy number alterations which are known to be linked with diseases that are associated with the symptoms the patient has.
  • Fusion proteins are useful for generating antibodies against PAKl polypeptides and for use in various assay systems. For example, fusion proteins can be used to identify proteins which interact with portions of PAKl polypeptides. Protein affinity chromatography or library-based assays for protein-protein interactions, such as> the yeast two-hybrid or phage display systems, can be used for this pu ⁇ ose. Such methods are well known in the art and also can be used as drug screens.
  • a PAKl fusion protein comprises two polypeptide segments fused together by means of a peptide bond.
  • the first polypeptide segment can comprise at least 54, 75, 100, 125, 139, 150, 175, 200, 225, 250, 275, 300, 325 or 350 contiguous amino acids of SEQ ID NO: 2 or of a biologically active variant, such as those described above.
  • the first polypeptide segment also can comprise full-length PAKl. , .
  • the second polypeptide segment can be a full-length protein or a protein fragment.
  • Proteins commonly used in fusion protein construction include, but are not limited to ⁇ -galactosidase, ⁇ - glucuronidase, green fluorescent protein (GFP), autofluorescent proteins, including blue fluorescent protein (BFP), glutathione-S-transferase (GST), luciferase, horseradish peroxidase (HRP), and chloramphenicol acetylfransferase (CAT).
  • epitope tags are used in fusion protein constructions, including histidine (His) tags, FLAG tags, influenza hemagglutinin (HA) tags, Myc tags, VSV-G tags, and thioredoxin (Trx) tags.
  • Other fusion constructions can include maltose binding protein (MBP), S-tag, Lex a DNA binding domain (DBD) fusions, GAL4 DNA binding domain fusions, and he ⁇ es .simplex virus (HSV) BP16 protein fusions.
  • a fusion protein also can be engineered to contain a cleavage site located adjacent to the PAKl .
  • a naturally occurring PAKl polynucleotide can be isolated free of other cellular components such as membrane components, proteins, and lipids.
  • Polynucleotides can be made by a cell and isolated using standard nucleic acid purification techniques, or synthesized using an amplification technique, such as the polymerase chain reaction (PCR), or by using an automatic synthesizer. Methods for isolating polynucleotides are routine and are known in the art. Any such technique for obtaining a polynucleotide can be used to obtain isolated PAKl polynucleotides. For example, restriction enzymes and probes can be used to isolate polynucleotide fragments which comprise PAKl nucleotide sequences. Isolated polynucleotides are in preparations which are free or at least 70, 80, or 90% free of other molecules.
  • PAKl cDNA molecules can be made with standard molecular biology techniques, using PAKl mRNA as a template. PAKl cDNA molecules can thereafter be replicated using molecular biology techniques known in the art. An amplification technique, such as PCR, can be used to obtain additional copies of polynucleotides of the invention, using either human genomic DNA or cDNA as a template.
  • PAKl polynucleotides can be synthesized using synthetic chemistry techniques to synthesizes PAKl polynucleotides.
  • the degeneracy of the genetic code allows alternate nucleotide sequences to be synthesized which will encode PAKl having, for example, an amino acid sequence shown in SEQ ID NO: 2 or a biologically active variant thereof.
  • PCR-based methods can be used to extend nucleic acid sequences encoding human PAKl, for example to detect upstream sequences of PAKl gene such as promoters and regulatory elements.
  • restriction-site PCR uses universal primers to retrieve unknown sequence adjacent to a known locus. Genomic DNA is first amplified in the presence of a primer to a linker sequence and a primer specific to the known region. The amplified sequences are then subjected to a second round of PCR with the same linker primer and another specific primer internal to the first one. Products of each round of PCR are transcribed. with an appropriate RNA polymerase and sequenced using reverse transcriptase.
  • Inverse PCR also can be used to amplify or extend sequences using divergent primers based on a known region.
  • Primers can be designed using comr ercially available software, such as OLIGO 4.06 Primer Analysis software (National Biosciemces Inc., Madison, Minn.), to be 22-30 nucleotides in length, to have a GC content of 50% or more, and to anneal to the target sequence at temperatures about 68-72°C.
  • the method uses several restriction enzymes to generate a suitable fragment in the known region of a gene. The fragment is then circularized by intramolecular ligation and used as a PCR template.
  • capture PCR which involves PCR amplification of DNA fragments adjacent to a known sequence in human aud yeast artificial chromosome DNA.
  • multiple restriction enzyme digestions and ligations also can be used to place an engineered double-stranded sequence into an unkno vn fragment of the DNA molecule before performing PCR.
  • Randomly-primed libraries are preferable, in that they will contain more sequences which contain the 5' regions of genes.
  • XJse of a randomly primed library may be especially preferable for situations in which an oligo d(T) library does not yield a full-length cDNA.
  • Genomic libraries can be useful for extension of sequence into 5' non-transcribed regulatory regions.
  • capillary electrophoresis systems can be used to analyze the size or confirm the nucleotide sequence of PCR or sequencing products.
  • capillary sequencing can employ flowable polymers for electrophoretic separation, four different fluorescent dyes (one for each nucleotide) which are laser activated, and detection of the emitted wavelengths by a charge coupled device camera.
  • Output/light intensity can be converted to electrical signal using appropriate equipment and software (e.g., GENOTYPER and Sequence NAVIGATOR, Perkin Elmer), and the entire process from loading of samples to comp iter analysis and electronic data display can be computer controlled.
  • Capillary electrophoresis is especially preferable for the sequencing of small pieces of DNA which might be present in limited amounts in a particular sample. ' •
  • PAKl can be obtained, for example, by purification from human cells, by expression of PAKl polynucleotides, or by direct chemical synthesis.
  • PAKl can be purified from any human cell which expresses the enzyme, including those which have been transfected with expression constructs which express PA--K1.
  • a purified PAKl is separated from other compounds which normally associate with PAKl in the cell, such as certain proteins, carbohydrates, or lipids, using methods well-known in the art. Such methods include, but are not limited to, size exclusion chromatography, ammonium sulfate fractionation, ion exchange chromatography, affinity chromatography, and preparative gel electrophoresis.
  • PAKl polynucleotides can be inserted into an expression vector which contains the necessary elements for the transcription and translation of the inserted coding sequence.
  • Methods which are well known to those skilled in the art can be used to construct expression vectors containing sequences encoding PAKl and appropriate transc riptional and translational control elements. These methods include in vitro recombinant D ' -NA techniques, synthetic techniques, and in vivo genetic recombination.
  • a variety of expression vector/host systems can be utilized to contain and express sequences encoding PAKl. These include, but are not limited to, microorganisms, such as bacteria transformed with recombinant bacteriophage, ⁇ lasmid, or cosmid DNA-. expression vectors; yeast transformed with yeast expression vectors', insect cell systems infected with virus expression vectors (e.g., baculovirus), plant cell systems transformed with virus, expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or with b acterial expression vectors (e.g., Ti or pBR322 plasmids), or animal cell systems.
  • microorganisms such as bacteria transformed with recombinant bacteriophage, ⁇ lasmid, or cosmid DNA-. expression vectors
  • yeast transformed with yeast expression vectors' insect cell systems infected with virus expression vectors (e.g., baculovirus), plant cell systems transformed with virus, expression vectors (e.g.,
  • control elements or regulatory sequences are those non-translated regions of the vector - enhancers, promoters, 5' and 3' untranslated regions — which interact with host cellular proteins to carry out transcription and translation. Such elements can vary in their strength and specificity. Depending on the vector system and host utilized, any number of suitable transcription and translation elements, including constitutive and inducible promoters, can be used. For example, when cloning in bacterial systems, inducible promoters si-tch as the hybrid lacZ promoter of the BLUESCRIPT phagemid (Stratagene, LaJolla,, Calif.) or pSPORTl plasmid (Life Technologies) and the like can be used.
  • the baculovirus polyhedrin promoter can be used in insect cells. Promoters or enhancers derived from the genomes of plant cells (e.g., heat shock, RUBISCO, and storage protein genes) or from plant viruses (e.g., viral promoters or leader sequences) can be cloned into the vector. In mammalian cell systems, promoters from mammalian genes or from mammalian viruses are preferable. If it is necessary to generate a cell line that contains multiple copies of a nucleotide sequence encoding PAKl, vectors ba-sed on SV40 or EBV can be used with an appropriate selectable marker.
  • a number of expression vectors can be selected. For example, when a large quantity of PAKl is needed for the induction of antibodies, vectors which direct high level expression of fusion proteins that are readily purified can t>e used.
  • vectors include, but are not limited to, multifunctional E. coli cloning and expression vectors such as BLUESCRIPT (Stratagene).
  • BLUESCRIPT a sequence encoding PAKl can be ligated into the vector in frame with sequences for the amino-terminal Met and the subsequent 7 residues of ⁇ - galactosidase so that a hybrid protein is produced.
  • pDST vectors or pGEX vectors also can be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST).
  • GST glutathione S-transferase
  • fusion proteins are soluble and can easily be purified from lysed cells by adso ⁇ tion to glutathione-agarose beads followed by elution in the presence of free glutathione.
  • Proteins made in such systems can be designed to include heparin, thrombin, or factor Xa protease cleavage sites so that the cloned polypeptide of interest can be released from the GST moiety at will.
  • sequences encoding PAKl can be driven by any of a number of promoters.
  • viral promoters such as the 35S and 19S promoters of CaMV can be used alone or in combination with the omega leader sequence from TMV.
  • plant promoters such as the small subunit of RTUBISCO or heat shock promoters can be used. These constructs can be introduced into plant cells by direct DNA transformation or by pathogen-mediated transfection.
  • An insect system also can be used to express PAKl.
  • Autographa californica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes in Spodoptera frugiperda cells or in Trichoplusi ⁇ larvae.
  • Sequences encoding PAKl can be cloned into a non-essential region of the virus, such as the polyhedrin gene, and placed under control of the polyhedrin promoter.
  • Successful insertion of PAKl will render the polyhedrin gene inactive and produce recombinant virus lacking coat protein.
  • the recombinant viruses can then be used to infect S. frugiperda cells or Trichoplusia larvae in which PAKl can be expressed.
  • a number of viral-based expression systems can be used to express PAKl in mai ⁇ malian host cells.
  • sequences encoding PAKl can be ligated into an adenovirus transcription/translation complex comprising the late promoter and tripartite leader sequence. Insertion in a non-essential El or E3 region of the viral genome can be used to obtain a viable virus which is capable of expressing PAKl in infecte ⁇ l host cells [Engelhard, 1994)].
  • transcription enhancers such as the Rous sarcoma -virus (RSV) enhancer, can be used to increase expression in mammalian host cells.
  • RSV Rous sarcoma -virus
  • HACs Human artificial chromosomes
  • HACs Human artificial chromosomes
  • 6M to 10M are constructed and delivered to cells via conventional delivery methods (e.g., liposomes, polycationic amino polymers, or vesicles).
  • Specific initiation signals also can be used to achieve more efficient translation of sequences encoding PAKl. Such signals include the ATG initiation codon and adjacent sequences. In cases where sequences encoding PAKl, its initiation codon, ancl upstream sequences are inserted into the appropriate expression vector, no additional transcxiptional or franslational control signals may be needed.
  • exogenous franslational control signals (including the AT 3 initiation codon) should be provided.
  • the initiation codon should be in the conect reading frame to ensure translation of the entire insert.
  • Exogenous franslational elements and initiation codon. s can be of various origins, both natural and synthetic.
  • a host cell strain can be chosen for its ability to modulate the expression of the inserted sequences or to process the expressed PAKl in the desired fashion.
  • modifications of the polypeptide include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation, and acylation.
  • Post-translational processing which cleaves a "prepro" form of ⁇ the polypeptide also can be used to facilitate conect insertion, folding and/or function.
  • Different host cells which have specific cellular machinery and characteristic mechanisms for post-translational activities (e.g., CHO, HeLa, MDCK, HEK293, and WI38), are available from the Ameri can Type Culture Collection (ATCC; 10801 University Boulevard, Manassas, VA 20110-2209) and can be chosen to ensure the conect modification and processing of the foreign protein.
  • ATCC Ameri can Type Culture Collection
  • Stable expression is prefened for long-term, high-yield production of recombinant proteins.
  • cell lines which stably express PAKl can be transformed using expression vectors which can contain viral origins of replication and/or. endogenous expression elements and a selectable marker gene on the same or on a separate vector. Following the introduction of the vector, cells can be allowed to grow for 1-2 days in an enriched medium before they are switched to a selective medium. The pu ⁇ ose of the selectable marker is to confer resistance to selection, and its presence allows growth and recovery of cells which successfully express the introduced PAKl sequences. Resistant clones of stably transformed cells can be proliferated using tissue culture te-chniques appropriate to the cell type.
  • Any number of selection systems can be used to recover transformed cell lines. These include, but are not limited to, the he ⁇ es simplex virus thymidine kinase [Logan, (1984)] and adenine phosphoribosyltransferase [Wigler, (1977)] genes which can be employed in ti or aprf cells, respectively. Also, antimetabolite, antibiotic, or herbicide resistance can be used as the basis for selection.
  • dhfr confers resistance to methotrexate [Lowy, (1980)]
  • npt confers resistance to the aminoglycosides, neomycin and G-418 [Wigler, (1980)]
  • als and pat confer resistance to chlorsulfuron and phosphinotricin acetylfransferase, respectively Colbere- Garapin, 1981].
  • Additional selectable genes have been described.
  • trpB allows cells to utilize indole in place of tryptophan, or hisD, which allows cells to utilize histinol in place of histidine.
  • Visible markers such as anthocyanins, ⁇ -glucuronidase and its substrate G-TJS, and luciferase and its substrate luciferin, can be used to identify transformants and to qua-ntify the amount of transient or stable protein expression attributable to a specific vector system
  • marker gene expression suggests that a PAKl polynucleotide is also present, its presence and expression may need to be confirmed. For example, if a sequence encoding PAKl is inserted within a marker gene sequence, transformed cells containing sequences which encode PAKl can be identified by the absence of marker gene function. Alterna_tively, a marker gene can be placed in tandem with a sequence encoding PAKl under the control of " a single promoter. Expression of the marker gene in response to induction or selection usually indicates expression of PAKl polynucleotide.
  • host cells which contain a PAKl polynucleotide and which express PAKl can be identified by a variety of procedures known -to those of skill in the art. These procedures i-nclude, but are not limited to, DNA-DNA or DNA-RNA hybridizations and protein bioassay or immunoassay techniques which include membrane, solution, or chip-based technologies for the detection and/or quantification of nucleic acid or protein.
  • these procedures i-nclude, but are not limited to, DNA-DNA or DNA-RNA hybridizations and protein bioassay or immunoassay techniques which include membrane, solution, or chip-based technologies for the detection and/or quantification of nucleic acid or protein.
  • the presence of a polynucleotide sequence encoding PAKl can be detected by DNA-DNA or DNA-RNA hybridization or amplification using probes or fragments or fragments of polynucleotides encoding PAKl.
  • Nucleic acid amplification-based assays involve the use of oligonu
  • a variety of protocols for detecting and measuring the expression of PAKl, using either polyclonal or monoclonal antibodies specific for the polypeptide, are known in the art. Examples include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescence activated cell sorting (FACS).
  • ELISA enzyme-linked immunosorbent assay
  • RIA radioimmunoassay
  • FACS fluorescence activated cell sorting
  • a two-site, monoclonal-based immunoassay using monoclonal antibodies reactive to two non-interfering epitopes on PAKl can be used, or a competitive binding assay can be employed.
  • labels and conjugation techniques are known by those skilled in the art and can be used in various nucleic acid and amino acid assays.
  • Means for producing labeled hybridization or PCR probes for detecting sequences related to polynucleotides encoding PAKl ixi clude oligolabeling, nick translation, end-labeling, or PCR amplification using a labeled nucleotide.
  • sequences encoding PAKl can be cloned into a vector for the production of an mRNA probe.
  • RNA probes are known in the art, are commercially available, and can be used to synthesize RNA probes in vitro by addition of labeled nucleotides and an appropriate RNA polymerase such as T7, T3, or SP6. These procedures can be conducted using a variety of commercially available kits (Amersham Pharmacia Biotech, Promega, and US Biochemical). Suitable reporter molecules or labels which can be used for ease of detection include radionuclides, enzymes, and fluorescent, chemiluminescent, or chromogenic agents, as well as substrates, cofactors, inhibitors, magnetic particles, and the like.
  • Host cells transformed with PAKl polynucleotides can be cultured under conditions suitable for the expression and recovery of the protein from cell culture.
  • the polypeptide produced by a transformed cell can be secreted or contained intracellularly depending on the sequence and/or the vector used.
  • expression vectors containing PAKl polynucleotides can be designed to contain signal sequences which direct secretion of soluble PAKl through a prokaryotic or eukaryotic cell membrane or which direct the membrane insertion of membrane-bound PAKl.
  • purification facilitating domains include, but are not limited to, metal chelating peptides such as histidine-tryptophan modules that allow purification on immobilized metals, protein A domains that allow purification on immobilized immunoglobulin, and the domain utilized in the FLAGS extension/affinity purification system (Immunex Co ⁇ ., Seattle, Wash.).
  • cleavable linker sequences such as those specific for Factor XA or enterokinase (Invitrogen, San Diego, CA) between the purification domain and PAKl also can be used to facilitate purification.
  • One such expression vector provides for expression of a fusion protein containing PAKl and 6 histidine residues preceding a thioredoxin or an enterokinase cleavage site. The histidine residues facilitate purification by. -MAC (immobilized metal ion affinity chromatography) Maddox, (1983)], while the enterokinase cleavage site provides a means for purifying PAKl from the fusion protein [Porath, (1992)].
  • Sequences encoding PAKl can be synthesized, in whole or in part, using chemical methods well known in the art.
  • PAKl itself can be produced using chemical methods to synthesize its amino acid sequence, such as by direct peptide synthesis using solid-phase techniques. Protein synthesis can either be performed using manual teclmiques or by automation. Automated synthesis can be achieved, for example, using Applied Biosystems 431 A Peptide Synthesizer (Perkin Elmer).
  • fragments of PAKl can be separately synthesized and combined using chemical methods to produce a full-length molecule.
  • the newly synthesized peptide can be substantially purified by preparative high performance liquid chromatography.
  • the composition of a synthetic PAKl " can be confirmed by amino acid analysis or sequencing. Additionally, any portion of the amino acid sequence of PAKl can be altered during direct synthesis and/or combined using chemical methods with sequences from other proteins to produce a variant polypeptide or a fusion protein. Production of Altered Polypeptides
  • codons prefened by a particular prokaryotic or eukaryotic host can be selected to increase the rate of protein expression or to produce an RNA transcript having desirable properties, such as a half-life which is longer than that of a transcript generated from the naturally occurring sequence.
  • nucleotide sequences refereed to herein can be engineered using methods generally known in the art to alter PAKl polynucleotides for a variety of reasons, including but not limited to, alterations which modify the cloning, processing, and/or expression of the polypeptide or mRNA product.
  • DNA shuffling by random fragmentation and PCR reassembly of gene fragments and synthetic oligonucleotides can be used to engineer the nucleotide sequences.
  • site- directed mutagenesis can be used to insert new restriction sites, alter glycosylation patterns, change codon preference, produce splice variants, introduce mutations, and so forth.
  • PAKl analogs are variants having an amino acid sequence that is a mutation of the amino acid sequence disclosed herein.
  • Another general class of PAKl analogs is provided by anti-idiotype antibodies, and fragments thereof, as described below.
  • recombinant antibodies comprismg anti-idiotype variable domains can be used as analogs (see, for example, [Monfardini et al., (1996)]). Since the variable domains of anti-idiotype PAKl antibodies mimic PAKl, these domains can provide PAKl enzymatic activity.
  • Methods of producing anti-idiotypic catalytic antibodies are known to those of skill in the art [Joron et al., (1992), Friboulet et al. (1994), Avalle et al., (1998)].
  • Any type of antibody known in the art can be generated to bind specifically to an epitope of PAKl.
  • Antibody as used herein includes intact immunoglobulin molecules, as well as fragments thereof, such as Fab, F(ab') 2 , and Fv, which are capable of binding an epitope of PAKl.
  • Fab fragments thereof
  • F(ab') 2 fragments thereof
  • Fv fragments thereof
  • epitopes which involve non-contiguous amino acids may require more, e.g., at least 15, 25, or 50 amino acid.
  • An antibody which specifically binds to an epitope of PAKl can be used therapeutically, as well as in immunochemical assays, such as Western blots, ELISAs, radioimmunoassays, immuno- histochemical assays, immunoprecipitations, -or other immunochemical assays known in the art.
  • immunochemical assays such as Western blots, ELISAs, radioimmunoassays, immuno- histochemical assays, immunoprecipitations, -or other immunochemical assays known in the art.
  • Various immunoassays can be used to identify antibodies having the desired specificity. Numerous protocols for competitive binding or immunoradiometric assays are well known in the art. Such immunoassays typically involve the measurement of complex formation between an immunogen and an antibody which specifically binds to the PAKl immunogen.
  • an antibody which specifically binds to PAKl provides a detection signal at least 5-, 10-, or 20-fold higher than a detection signal provided with other proteins when used in an immunochemical assay.
  • antibodies which specifically bind to PAKl do not detect other proteins in immunochemical assays and can immunoprecipitate PAKl from solution.
  • PAKl can be used to immunize a mammal, such as a mouse, rat, rabbit, guinea pig, monkey, or human, to produce polyclonal antibodies. If desired, PAKl can be conjugated to a canier protein, such as bovine serum albumin, thyroglobulin, and keyhole limpet hemocyanin. Depending on the host species, various adjuvants can be used to increase the immunological response.
  • a mammal such as a mouse, rat, rabbit, guinea pig, monkey, or human
  • PAKl can be conjugated to a canier protein, such as bovine serum albumin, thyroglobulin, and keyhole limpet hemocyanin.
  • various adjuvants can be used to increase the immunological response.
  • Such adjuvants include, but are not limited to, Freund's adjuvant, mineral gels (e.g., aluminum hydroxide), and surface active substances (e.g., lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyaiiin, and dinitrophenol).
  • BCG Bacilli Calmette-Guerin
  • Corynebacterium parvum are especially useful.
  • Monoclonal antibodies which specifically bind to PAKl can be prepared using any technique which provides for the production of antibody molecules by continuous cell lines in culture. These techniques include, but are not limited to, the hybridoma technique, the human B-cell hybridoma technique, and the EBV-hybridoma technique [Roberge, (1995)].
  • chimeric antibodies the splicing of mouse antibody genes to human antibody genes to obtain a molecule with appropriate antigen specificity and biological activity
  • Monoclonal and other antibodies also can be "humanized” to prevent a patient from mounting an immune response against the antibody when it is used therapeutically.
  • Such antibodies may be sufficiently similar in sequence to human antibodies to be used directly in therapy or may require alteration of a few key residues. Sequence differences between rodent antibodies and human sequences can be minimized by replacing residues which differ from those in the human sequences by site directed mutagenesis of individual residues or by grating of entire complementarity determining regions.
  • Antibodies which specifically bind to PAKl can contain antigen binding sites which are either partially or fully humanized, as disclosed in U.S. 5,565,332.
  • single chain antibodies can be adapted using methods known in the art to produce single chain antibodies which specifically bind to PAKl.
  • Antibodies with related specificity, but of distinct idiotypic composition can be generated by chain shuffling from random combinatorial immunoglobin libraries.
  • Single-chain antibodies also can be constructed using a DNA amplification method, such as PCR, using hybridoma cDNA as a template.
  • Single-chain antibodies can be mono- or bispecific, and can be bivalent or tetravalent. Construction of tetravalent, bispecific single-chain antibodies is taught.
  • a nucleotide sequence encoding a single-chain antibody can be constr cted using manual or automated nucleotide synthesis, cloned into an expression construct using standard recombinant DNA methods, and introduced into a cell to express the coding sequence, as described below.
  • single-chain antibodies can be produced directly using, for example, filamentous phage technology.
  • Antibodies which specifically bind to PAKl also can be produced by inducing in vivo production in the lymphocyte population or by screening immunoglobulin libraries or panels of highly specific binding reagents.
  • Other types of antibodies can be constructed and used therapeutically in methods of the invention.
  • chimeric antibodies can be constructed as disclosed in WO 93/03151.
  • Antibodies according to the invention can be purified by methods well known in the art. For example, antibodies can be affinity purified by passage over a column to which PAKl is bound. The bound antibodies can then be eluted from the column using a buffer with a high salt concentration. . '
  • Antisense oligonucleotides are nucleotide sequences which are complementary to a specific DNA or RNA sequence. Once introduced into a cell,. the complementary nucleotides combine with natural sequences produced by the cell to form complexes and block either transcription or translation.
  • an antisense oligonucleotide is at least 11 nucleotides in length, but can be at least 12, 15, 20, 25, 30, 35, 40, 45, or 50 or more nucleotides long. Longer sequences also can be used.
  • Antisense oligonucleotide molecules can be provided in a DNA construct and introduced into a cell as described above to decrease the level of PAKl gene products in the cell.
  • Antisense oligonucleotides can be deoxyribonucleotides, ribonucleotides, or a combination of both. Oligonucleotides can be synthesized manually or by an automated synthesizer, by covalently linking the 5' end of one nucleotide with the 3' end of another nucleotide with non-phosphodiester internucleotide linkages such alkylphosphonates, phosphorothioates, phosphorodithioates, alkylphosphonothioates, alkylphosphonates, phosphoramidates, phosphate esters, carbamates, acetamidate, carboxymethyl esters, carbonates, and phosphate triesters.
  • Modifications of PAKl gene expression can be obtained by designing antisense oligonucleotides which will form duplexes to the confrol, 5', or regulatory regions of the PAKl gene. Oligonucleotides derived from the transcription initiation site, e.g., between positions -10 and +10 from the start site, are prefened. Similarly, inhibition can be achieved using "triple helix" base-pairing methodology. Triple helix pairing is useful because it causes inhibition of the ability of the double helix to open sufficiently for the binding of polymerases, transcription factors, or chaperons. Therapeutic advances using triplex DNA have been described in the literature [Nicholls, (1993)]. An antisense oligonucleotide also can be designed to block translation of mRNA by preventing the transcript from binding to ribosomes.
  • Antisense oligonucleotides which comprise, for example, 2, 3, 4, or 5 or more stretches of contiguous nucleotides which are precisely complementary to a PAKl polynucleotide, each separated by a stretch of contiguous nucleotides which are not complementary to adjacent PAKl nucleotides, can provide sufficient targeting specificity for PAKl mRNA.
  • each stretch of complementary contiguous nucleotides is at least 4, 5, 6, 7, or 8 or more nucleotides in length.
  • Non- complementary intervening sequences are preferably 1, 2, 3, or 4 nucleotides in length.
  • One skilled in the art can easily use the calculated melting point of an antisense-sense pair to determine the degree of mismatching which will be tolerated between a particular antisense oligonucleotide and a particular PAKl polynucleotide sequence.
  • Antisense oligonucleotides can be modified without affecting their ability to hybridize to a PAKl polynucleotide. These modifications can be internal or at one or both ends of the antisense molecule.
  • internucleoside phosphate linkages can be modified by adding cholesteryl or diamine moieties with varying numbers of carbon residues between the amino groups and terminal ribose.
  • Modified bases and/or sugars such as arabinose instead of ribose, or a 3', 5 '-substituted oligonucleotide in which the 3' hydroxyl group or the 5' phosphate group are substituted, also can be employed in a modified antisense oligonucleotide.
  • modified oligonucleotides can be prepared by methods well known in the art. Ribozymes
  • Ribozymes are RNA molecules with catalytic activity [Uhlmann, (1987)]. Ribozymes can be used to inhibit gene function by cleaving an RNA sequence, as is known in the art. The mechanism of ribozyme action involves sequence-specific hybridization of the ribozyme molecule to complementary target RNA, followed by endonucleolytic cleavage. Examples include engineered hammerhead motif ribozyme molecules that caii' specifically and efficiently catalyze endonucleolytic cleavage of specific nucleotide sequences.
  • the coding sequence of a PAKl polynucleotide can be used to generate ribozymes which will specifically bind to mRNA transcribed from a PAKl polynucleotide.
  • Methods of designing and constructing ribozymes which can cleave other RNA molecules in trans in a highly sequence specific manner have been developed and described in the art.
  • the cleavage activity of ribozymes can be targeted to specific RNAs by engineering a discrete "hybridization" region into the ribozyme.
  • the hybridization region contains a sequence complementary to the target RNA and thus specifically hybridizes with the target RNA.
  • Specific ribozyme cleavage sites within a PAKl RNA target can be identified by scanning the target molecule for ribozyme cleavage sites which include the following sequences: GUA, GUU, and GUC. Once identified, short RNA sequences of between 15 and 20 ribonucleotides conesponding to the region of the target RNA containing the cleavage site can be evaluated for secondary structural features which may render the target inoperable. Suitability of candidate PAKl RNA targets also can be evaluated by testing accessibility to hybridization with complementary oligonucleotides using ribonuclease protection assays. The nucleotide sequences shown in SEQ ID NO: 1 and its complement provide sources of suitable hybridization region sequences.
  • hybridizing and cleavage regions of the ribozyme can be integrally related such that upon hybridizing to the target RNA through the complementary regions, the catalytic region of the ribozyme can cleave the target.
  • Ribozymes can be introduced into cells as part of a DNA construct. Mechanical methods, such as microinjection, liposome-mediated transfection, electroporation, or calcium phosphate precipitation, can be used to introduce a ribozyme-containing DNA construct into cells in which it is desired to decrease PAKl expression. Alternatively, if it is desired that the cells stably retain the DNA construct, the construct can be supplied on a plasmid and maintained as a separate element or integrated into the genome of the cells, as is known in the art.
  • a ribozyme-encoding DNA construct can include transcriptional regulatory elements, such as a promoter element, an enhancer or UAS element, and a transcriptional terminator signal, for controlling transcription of ribozymes in the cells (U.S. 5,641,673). Ribozymes also can be engineered to provide an additional level of regulation, so that destruction of rnKNA occurs only when both a ribozyme and a target gene are induced in the cells.
  • Regulators as used herein refer to compounds that affect the activity of PAKl in vivo and/or in vitro. Regulators can be agonists and antagonists of PAKl polypeptide and can be compounds that exert their effect on the PAKl activity via the enzymatic activity, expression, post-translational modifications or by other means.
  • Agonists of PAKl are molecules which, when bound to PAKl, increase or prolong the activity of PAKl.
  • Agonists of PAKl include proteins, nucleic acids, carbohydrates, small molecules, or any other molecule which activate PAKl.
  • Antagonists of PAKl are molecules which, when bound to PAKl, decrease the amount or the duration of the activity of PAKl. Antagonists include proteins, nucleic acids, carbohydrates, antibodies, small molecules, or any other molecule which decrease the activity of PA l .
  • modulate refers to a change in the activity of PAKl polypeptide. For example, modulation may cause an increase or a decrease in enzymatic activity, binding characteristics, or any other biological, functional, or immunological properties of PAKl.
  • the terms “specific binding” or “specifically binding” refer to that interaction between a protein or peptide and an agonist, an antibody, or an antagonist. The interaction is dependent upon the presence of a particular structure of the protein recognized by the binding molecule (i.e., the antigenic determinant or epitope). For example, if an antibody is specific for epitope "A" the presence of a polypeptide containing ' the epitope A, or the presence of free unlabeled A, in a reaction containing free labeled A and the antibody will reduce the amount of labeled A that binds to the antibody.
  • the invention provides methods (also refened to herein as "screening assays") for identifying compounds which can be used for the treatment of diseases related to PAKl.
  • the methods entail the identification of candidate or test compounds or agents (e.g., peptides, peptidomimetics, small molecules or other molecules) which bind to PAKl and/or have a stimulatory or inhibitory effect on the biological activity of PAKl or its expression and then determining which of these compounds have an effect on symptoms or diseases related to PAKl in an in vivo assay.
  • candidate or test compounds or agents e.g., peptides, peptidomimetics, small molecules or other molecules
  • Candidate or test compounds or agents which bind to PAKl and/or have a stimulatory or inhibitory effect on the activity or the expression of PAKl are identified either in assays that, employ cells which express PAKl (cell-based assays) or in assays with isolated PAKl (cell-free assays).
  • the various assays can employ a variety of variants of PAKl (e.g., full-length PAKl, a biologically active fragment of PAKl, or a fusion protein which includes all or a portion of PAKl).
  • PAKl can be derived from any suitable mammalian species (e.g., human PAKl, rat PAKl or murine PAKl).
  • the assay can be a binding assay entailing direct or indirect measurement of the binding of a test compound or a known PAKl ligand to PAKl.
  • the assay can also be an activity assay entailing direct or indirect measurement of the activity of PAKl.
  • the assay can also be an expression assay entailing direct or indirect measurement of the expression of PAKl mRNA or PAKl protein.
  • the various screening assays are combined with an in vivo assay entailing measuring the effect of the test compound on the symptoms of diseases related to PAKl.
  • the present invention includes biochemical, cell free assays that allow the identification of inhibitors and agonists of kinases suitable as lead structures for pharmacological drug development.
  • Such assays involve contacting a form of PAKl (e.g., full-length PAKl, a biologically active fragment of PAKl, or a fusion protein comprising all or a portion of PAKl) with a test compound and determining the ability of the test compound to act as an antagonist (preferably) or an agonist of the enzymatic activity of PAK 1.
  • PAKl e.g., full-length PAKl, a biologically active fragment of PAKl, or a fusion protein comprising all or a portion of PAKl
  • PAKl molecules of the present invention can be measured using a variety of assays that measure PAKl activity.
  • PAKl enzyme activity can be assessed by a standard in vitro kinase assay.
  • PAKl can be used in substantial and specific assays related to kinases.
  • assays involve any of the known kinase functions or activities or properties useful for diagnosis and treatment of kinase- related conditions that are specific for the subfamily of kinases that the one of the present invention belongs to, particularly in cells and tissues that express the kinase.
  • PAKl is also useful in drug screening assays, in cell-based or cell-free systems.
  • PAKl can be used to identify compounds that modulate kinase activity of the protein in its natural state or an altered form that causes a specific disease or. pathology associated with the kinase.
  • the present invention also relates to a method of screening compounds having inhibitory activity of kinase activity of the proteins of the present invention.
  • This screening method consists of two steps. First, PAKl is caused to contact a substrate to be phosphorylated by this protein in the presence of a test compound to detect the kinase activity of the protein of the present invention. Second, the kinase activity detected in step (a) is compared with that detected in the absence of the test compound, and a compound that lowers the kinase activity of the protein of the present invention is selected.
  • the kinase activity of PAKl can be detected, for example, by adding ATP having radioactively labeled phosphate to the reaction system containing the protein of the present invention and the substrate and measuring the radioactivity of the phosphate attached to the substrate.
  • Both, PAKl and appropriate variants and fragments can be used in high-throughput screens to assay candidate compounds for the ability to modulate the kinase activity. These compounds can be further screened against a functional kinase to determine the effect of the compound on the kinase activity. Further, these compounds can be tested in animal or invertebrate systems to determine activity/effectiveness. Compounds can be identified that activate (agonist) or inactivate (antagonist) the kinase to a desired degree.
  • PAKl can be used to screen a compound for the ability to stimulate or inhibit interaction between the kinase protein and a molecule that normally interacts with the kinase protein, e.g. a substrate or a component of the signal pathway, that the kinase protein normally interacts (for example, another kinase).
  • a molecule that normally interacts with the kinase protein e.g. a substrate or a component of the signal pathway, that the kinase protein normally interacts (for example, another kinase).
  • Such assays typically include the steps of combining the kinase protein with a candidate compound under conditions that allow the kinase protein, or fragment, to interact with the target molecule, and to detect the formation of a complex between the protein and the target or to detect the biochemical consequence of the interaction with the kinase protein and the target, such as any of the associated effects of signal transduction such as protein phosphorylation, cAMP turnover, and adenylate cyclase activation, etc.
  • the invention further includes other end point assays to identify compounds that modulate (stimulate or inhibit) PAKl kinase activity.
  • the assays typically involve an assay of events in the signal transduction pathway that indicate kinase activity.
  • the phosphorylation of a substrate, activation of a protein, a change in the expression of genes that are up- or down-regulated in response to the kinase protein dependent signal cascade can be assayed.
  • any of the biological or biochemical functions mediated by the kinase can be used as an endpoint assay. These include all of the biochemical or biochemical/biological events described herein, in the references cited herein, inco ⁇ orated by reference for these endpoint assay targets, and other functions known to those of ordinary skill in the art.
  • Binding and/or activating compounds can also be screened by using chimeric kinase proteins in which the amino terminal extracellular domain, or parts thereof, the entire transmembrane domain or subregions, such as any of the seven transmembrane segments or any of the intracellular or extracellular loops and the carboxy terminal intracellular domain, or parts thereof, can be replaced by heterologous domains or subregions.
  • a substrate-binding region can be used that interacts with a different substrate then that which is recognized by the native kinase. Accordingly, a different set of signal transduction components is available as an end-point assay for activation. This allows for assays to be performed in other than the specific host cell from which the kinase is derived.
  • PAKl is also useful in competition binding assays in methods designed to discover compounds that interact with the kinase (e.g. binding partners and/or ligands).
  • a compound is exposed to a kinase polypeptide under conditions that allow the compound to bind or to otherwise interact with the polypeptide.
  • Soluble kinase polypeptide is also added to the mixture. If the test compound interacts with the soluble kinase polypeptide, -it decreases the amount of complex formed or activity from the kinase target. This type of assay is particularly useful in cases in which compounds are sought that interact with specific regions of the kinase.
  • Agents that modulate PAKl can be identified using one or more of the above assays, alone or in combination. It is generally preferable to use a cell-based or cell free system first and then confirm activity in an animal or other model system. Such model systems are well known in the art and can readily be employed in this context.
  • Test compounds used for this screening methods are not particularly limited and are generally low- molecular-weight compounds, proteins (including antibodies), peptides, etc. Test compounds are either artificially synthesized or natural.
  • an antibody is defined in terms consistent with that recognized within the art: they are multi-subunit proteins produced by a mammalian organism in response to an antigen challenge.
  • the antibodies of the present invention include polyclonal antibodies and monoclonal antibodies, as well as fragments of such antibodies, including, but not limited to, Fab or F(ab').sub.2, and Fv fragments.
  • an isolated peptide is used as an immunogen and is administered to a mammalian organism, such as a rat, rabbit or mouse.
  • a mammalian organism such as a rat, rabbit or mouse.
  • the full-length protein, an antigenic peptide fragment or a fusion protein can be used.
  • Particularly important fragments are those covering functional domains, such as the domains identified in FIG. 2, and domain of sequence homology or divergence amongst the family, such as those that can readily be identified using protein alignment methods and as presented in the Figures.
  • Antibodies are preferably prepared from regions or discrete fragments of the kinase proteins. Antibodies can be prepared from any region of the peptide as described herein. However, prefened regions will include those involved in function/a ⁇ ?tivity and/or kinase/binding partner interaction.
  • Modulators of kinase protein activity identified according to these drug screening assays can be used to treat a subject with a disorder mediated by the kinase pathway, by treating cells or tissues that express the kinase.
  • This invention further pertains to novel agents identified by the above-described screening assays. Accordingly, it is within the scope of this invention to further use an agent identified as described herein in an appropriate animal model.
  • an agent identified as described herein e.g., a kinase-modulating agent, an antisense kinase nucleic acid molecule, a kinase-specif ⁇ c antibody, or a kinase-binding partner
  • an agent identified as described herein can be used in an animal or other model to determine the efficacy, toxicity, or side effects of treatment with such an agent.
  • an agent identified as described herein can be used in an animal or other model to determine the mechanism of action of such an agent.
  • this invention pertains to uses of novel agents identified by the above- described screening assays for treatments as described herein.
  • PAKl proteins are also useful to provide a target for diagnosing a disease or predisposition to disease mediated by the peptide. Accordingly, the invention provides methods for detecting the presence, or levels of, the protein (or encoding mRNA) in a cell, tissue, or organism-
  • a biological sample includes tissues, cells and biological fluids isolated from a subject, as well as tissues, cells and fluids present within a subject..
  • the peptides of the present invention also provide targets for diagnosing active protein activity, disease, or predisposition to disease, in a patient having a variant peptide, particularly activities and conditions that are known for other members of the family of proteins to which the present one belongs.
  • the peptide can be isolated from a biological sample and assayed for the presence of a genetic mutation that results in abenant peptide. This includes amino acid substitution, deletion, insertion, reanangement, (as the result of abenant splicing events), and inappropriate post- franslational modification.
  • Analytic methods include altered electrophoretic mobility, altered tryptic peptide digest, altered kinase activity in cell-based or cell-free assay, alteration in substrate or antibody-binding pattern, altered isoelectric point, direct amino acid sequencing, and any other of the known assay techniques useful for detecting mutations in a protein.
  • the diseases for which detection of kinase genes in a sample could be diagnostic include diseases in which kinase nucleic acid (DNA and/or R A) is amplified in comparison to normal cells.
  • amplification is meant increased numbers of kinase DNA or RNA in a cell compared with normal cells.
  • kinases are typically found as single copy genes.
  • the chromosomal location of the kinase genes may be amplified, resulting in multiple copies of the gene, or amplification.
  • Gene amplification can lead to amplification of kinase RNA, or kinase RNA can be amplified in the absence of kinase DNA amplification.
  • In vitro techniques for detection of peptide include enzyme linked immunosorbent assays (ELISAs), Western blots, immunoprecipitations and immunofluorescence using a detection reagent, such as an antibody or protein binding agent.
  • a detection reagent such as an antibody or protein binding agent.
  • the peptide can be detected in vivo in a subject by introducing into the subject a labeled anti-peptide antibody or other types of detection agent.
  • the antibody can be labeled with a radioactive marker whose presence and location in a subject can be detected by standard imaging techniques.
  • Particxilarly useful are methods that detect the allelic variant of a peptide expressed in a subject and methods which detect fragments of a peptide in a sample.
  • Solution in vitro assays can be used to identify a PAKl substrate or inhibitor.
  • Solid phase systems can also be used to identify a substrate or irJ ribitor of a PAKl polypeptide.
  • a PAKl polypeptide or PAKl fusion protein can be immobilized onto the surface of a receptor chip of a commercially available biosensor instrument (BIACORE, Biacore AB; Uppsala, Sweden). Tbe use of this instrument is disclosed, for example, by [Karlsson, (1991), and Cunningham and Wells, (1993)].
  • a PAKl polypeptide or fusion protein is covalently attached, using amine or sulfhydryl chemistry, to dextran fibers that are attached to gold film within a flow cell.
  • a test sample is then passed through the cell. If a PAKl substrate ox inhibitor is present in the sample, it will bind to the immobilized polypeptide or fusion protein, caxising a change in the refractive index of the medium, which is detected as a change in surface plasmon resonance of the gold film.
  • This system allows the determination on- and off-rates, from which ' binding affinity can be calculated, and assessment of the stoichiometry of binding, as well as the kinetic effects of PAKl mutation.
  • the invention provides assays for screening candidate or test compounds which bind to or modulate the activity of PAKl.
  • assays can employ full-length PAKl, a biologically active fragment of PAKl, or a fusion protein which includes all or a portion of PAKl.
  • the test compound can be obtained by any suitable means, e.g., from conventional compound libraries.
  • Determining the ability of the test compound to ' modulate the activity of PAKl can be accomplished, for example, by determining the ability of PAKl to bind to or interact with a target molecule.
  • the target molecule can be a molecule with which PAKl binds or interacts with in nature.
  • the target molecule can be a component of a signal transduction pathway which facilitates transduction of an extracellular signal.
  • the target PAKl molecule can be, for example, a second intracellular protein which has catalytic activity or a protein which facilitates the association of downstream signaling molecules with PAKl ' .
  • Determining the ability of PAKl to bind to or interact with a target molecule can be accomplished by one of the methods described above for determining direct binding. In one embodiment, determining the ability of a polypeptide of the invention to bind to or interact with a target molecule can be accomplished by determining the activity of the target molecule.
  • the activity of the target molecule can be determined by detecting induction of a cellular second messenger of the target (e.g., intracellular Ca 2+ , diacylglycerol, ff 3 , etc.), detecting catalytic/- enzymatic activity of the target on an appropriate substrate, detecting the induction of a reporter gene (e.g., a regulatory element that is responsive to a polypeptide of the invention operably linked to a nucleic acid encoding a detectable marker, e.g., luciferase), or detecting a cellular response.
  • a reporter gene e.g., a regulatory element that is responsive to a polypeptide of the invention operably linked to a nucleic acid encoding a detectable marker, e.g., luciferase
  • PAKl or a PAKl target molecule
  • Binding of a test compound to PAKl, or interaction of PAKl with a target molecule in the presence and absence of a candidate compound can be accomplished in any vessel suitable for containing the reactants. Examples of such vessels include microtitre plates, test tubes, and micro- centrifuge tubes.
  • a fusion protein can be provided which adds a domain that allows one or both of the proteins to be bound to a matrix.
  • glutathione-S-transferase (GST) fusion proteins or glutathione-S-transferase fusion proteins can be adsorbed onto glutathione sepharose beads (Sigma Chemical; St. Louis, Mo.) or glutathione derivatized microtitre plates, which are then combined with the test compound or the test compound and either the non-adsorbed target protein or PAKl, and the mixture incubated under conditions conducive to complex formation (e.g., at physiological conditions for salt and pH). Following incubation, the beads or microtitre plate wells are washed to remove any unbound components and complex formation is measured either directly or indirectly, for example, as described above. Alternatively, the complexes can be dissociated from the matrix, and the level of binding or activity of PAKl can be determined using standard techniques.
  • PAKl or its -target molecule can be immobilized utilizing conjugation of biotin and streptavidin.
  • Biotinylated polypeptide of the invention or target molecules can be prepared from biotin-NHS (N : hydro ⁇ .y-succinimide) using techniques well known in the art (e.g., biotinylation kit, Pierce Chemicals; Rockford, 111.), and immobilized in the wells of sfreptavidin-coated plates (Pierce Chemical).
  • antibodies reactive with PAKl or target molecules but which do not interfere with binding of the polypeptide of the invention to its target molecule can be derivatized to the wells of the plate, and unbound target or polypeptide of the invention trapped in the wells by antibody conjugation.
  • Methods for detecting such complexes include immunodetection of complexes using antibodies reactive wdth PAKl or target molecule, as well as enzyme-linked assays which rely on detecting an enzymatic activity associated with PAKl or target molecule.
  • Another technique for drug screening which may be used jprovides for high throughput screening of compounds having suitable binding affinity to the protein of interest as described in published PCT application WO84/03564.
  • large numbers of different small test compounds are synthesized on a solid substrate, such as plastic pins or some other surface.
  • the test compounds are reacted with PAKl, or fragments thereof, and washed.
  • Bound PAKl is then detected by methods well known in the art.
  • Purified PAKl can also be coated directly onto plates for use in the aforementioned drug screening techni-j_ues.
  • non-neutralizing antibodies can be used to capture the peptide and immobilize it on a solid support.
  • the screening assay can also involve monitoring the expression of PAKl .
  • regulators of expression of PAKl can be identified in a method in which a cell is contacted with a candidate compound and the expression of PAKl protein or mRNA an the cell is determined. The level of expression of PAKl protein or mRNA the presence of the candidate compound is compared to the level of expression of PAKl protein or mRNA in the absence of the candidate compound. The candidate compound can then be identified as a regulator of expression of PAKl based on this comparison. For example, when expression of PAKl protein or mRNA protein is greater (statistically significantly greater) in the presence of the candidate compound than in its absence, the candidate compound is identified as a stimulator of PAKl protein or mRNA expression.
  • tb-e candidate compound when expression of PAKl protein or mRNA is less (statistically significantly less) in the presence of the candidate compound than in its absence, tb-e candidate compound is identified as an inhibitor of PAKl protein or mRNA expression.
  • the level of PAKl protein or mRNA expression in the cells can be determined by methods described " below.
  • Such screening can be canied out either in a cell-free assay system or in an intact cell.
  • Any cell which expresses PAKl polynucleotide can be used in a cell-based assay system.
  • the PAKl polynucleotide can be naturally occurring in the cell or can be introduced using techniques such as those described above. Either a primary culture or an established cell line can be used.
  • the test compound is preferably a small molecule ⁇ vhich binds to and occupies the active site of PAKl polypeptide, thereby making the ligand binding site inaccessible to substrate such that normal biological activity is prevented.
  • Example s of such small molecules include, but are not limited to, small peptides or peptide-like molecule-s.
  • Potential ligands which bind to a polypeptide of the invention include, but are not limited to, .be natural ligands of known PAKl kinases and analogues or derivatives thereof.
  • either the test compound or the PAKl polypeptide can comprise a detectable label, such as a fluorescent, radioisotopic, chemiluminescent, or enzymatic label, such as horseradish peroxidase, alkaline phosphatase, or luciferase. Detection of a test compound which is bound to PAKl polypeptide can then be accomplished, for example, by direct counting of radio- emmission, by scintillation counting, or by determining conversion of a_n appropriate substrate to a detectable product. Alternatively, binding of a test compound to a PAKl polypeptide can be determined without labeling either of the interactants.
  • a detectable label such as a fluorescent, radioisotopic, chemiluminescent, or enzymatic label, such as horseradish peroxidase, alkaline phosphatase, or luciferase.
  • a microphysiometer can be used to detect binding of a test compound with a PAKl polypeptide.
  • a microphysiometer e.g., CytosensorTM
  • LAPS light-addressable potentiometric sensor
  • BIA Bimolecular Interaction Analysis
  • a PAKl -like polypeptide in yet another aspect of the invention, can be used as a "bait protein" in a two-hybrid assay or three-hybrid assay [Szabo, (1995); U.S. 5,283,317), to identify other proteins which bind to or interact with PAKl and modulate its activity.
  • the two-hybrid system is based on the modular nature of most transcription factors, which consist of separable DNA-binding and activation domains.
  • the assay utilizes two different DNA constructs.
  • polynucleotide encoding PAKl can be fused to a polynucleotide encoding the DNA binding domain of a known transcription factor (e.g., GAL-4).
  • a DNA sequence that encodes an unidentified protein (“prey" or "sample" can be fused to a polynucleotide that codes for the activation domain of the known transcription factor.
  • the DNA-binding and activation domains of the transcription factor are brought into close proximity. This proximity allows transcription of a xeporter gene (e.g., LacZ), which is operably linked to a transcriptional regulatory site responsive to the transcription factor. Expression of the reporter gene can be detected, and cell colonies containing the functional transcription factor can be isolated and used to obtain the DNA sequence encoding the protein which interacts with PAK 1.
  • a xeporter gene e.g., LacZ
  • either the PAKl (or polynucleotide) or the test compound can be bound to a solid support.
  • suitable solid supports include, but are not limited to, glass or plastic slides, tissue culture plates, microtiter wells, tubes, silicon chips, or particles such as beads (including, but not limited to, latex, polystyrene, or glass beads).
  • Any method known in the art can be used to attach PAKl -like polypeptide (or polynucleotide) or test compound to a solid support, including use of covalent and non-covalent linkages, passive abso ⁇ tion, or pairs of binding moieties attached respectively to the polypeptide (or polynucleotide) or test compound and the solid support.
  • Test compounds are preferably bound to the solid support in an anay, so that the location of individual test compounds can be tracked. Binding of a test compound to PAKl (or a polynucleotide encoding for PAKl) can be accomplished in any vessel suitable for containing the reactants. Examples of such vessels include microtiter plates, test tubes, and microcentrifuge tubes.
  • PAKl is a fusion protein comprising a domain that allows binding of PAKl to a solid support.
  • glutathione-S-transferase fusion proteins can be adsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis, Mo.) or glutathione derivatized microtiter plates, which are then combined with the test compound or the test compound and the non- adsorbed PAKl; the mixture is then incubated under conditions conducive to complex formation (e.g., at physiological conditions for salt and pH). Following incubation, the beads or microtiter plate wells are washed to remove any unbound components. Binding of the interactants can be determined either directly or indirectly, as described above. Alternatively, the complexes can be dissociated from the solid support before binding is determined.
  • PAKl or a polynucleotide encoding PAKl
  • test compound can be immobilized utilizing conjugation of biotin and streptavidin.
  • Biotinylated PAKl (or a polynucleotide encoding biotinylated PAKl) or test compounds can be prepared from biotin-NHS (N-hydroxysuccinimide) using techniques well known in the art (e.g., biotinylation kit, Pierce Chemicals, Rockford, 111.) and immobilized in the wells of sfreptavidin- coated plates (Pierce Chemical).
  • biotinylation kit Pierce Chemicals, Rockford, 111.
  • antibodies which specifically bind to PAKl, polynucleotide, or a test compound, but which do not interfere with a desired binding site, such as the active site of PAKl can be derivatized to the wells of the plate. Unbound target or protein can be trapped in the wells by antibody conjugation.
  • Methods for detecting such complexes include immunodetection of complexes using antibodies which specifically bind to PAKl polypeptide or test compound, enzyme-linked assays which rely on detecting an activity of PAKl polypeptide, and SDS gel electrophoresis under non-reducing conditions.
  • Any cell which comprises a PAKl polypeptide or polynucleotide can be used in a cell-based assay system.
  • a PAKl polynucleotide can be naturally occurring in the cell or can be introduced using techniques such as those described above. Binding of the test compound to PAKl or a polynucleotide encoding PAKl is determined as described above.
  • Test compounds can be tested for the ability to increase or decrease PAKl activity of a PAKl polypeptide.
  • the PAKl activity can be measured, for example, using methods described in the specific examples, below.
  • PAKl activity can be measured after contacting either a purified PAKl or an intact cell with a test compound.
  • a test compound which decreases PAKl activity by at least about 10, preferably about 50, more preferably about 75, 90, or 100% is identified as a potential agent for decreasing PAKl activity.
  • a test compound which increases PAKl activity by at least about 10, preferably about 50, more preferably about 75, 90, or 100% is identified as a potential agent for increasing PAKl activity.
  • test compounds which increase or decrease PAKl gene expression are identified.
  • the term "conelates with ex ression of a polynucleotide” indicates that the detection of the presence of nucleic acids, the same or related to a nucleic acid sequence encoding PAKl, by northern analysis or realtime PCR is indicative of the presence of nucleic acids encoding PAKl in a sample, and thereby conelates with expression of the transcript from the polynucleotide encoding PAKl.
  • microanay refers to an anay of distinct polynucleotides or oligonucleotides anayed on a substrate, such as paper, nylon or any other type of membrane, filter, chip, glass slide, or any other suitable solid support.
  • a PAKl polynucleotide is contacted with a test compound, and the expression of an RNA or polypeptide product of PAKl polynucleotide is determined.
  • the level of expression of appropriate mRNA or polypeptide in the presence of the test compound is compared to the level of expression of mRNA or polypeptide in the absence of the test compound.
  • the test compound can then be identified as a regulator of expression based on this comparison.
  • test compound when expression of mRNA or polypeptide is greater in the presence of the test compound than in its absence, the test compound is identified as a stimulator or enhancer of the mRNA or polypeptide expression.
  • test compound when expression of the mRNA or polypeptide is less in the presence of the test compound than in its absence, the test compound is identified as an inhibitor of the mRNA or polypeptide expression.
  • the level of PAKl mRNA or polypeptide expression in the cells can be determined by methods well known in the art for detecting mRNA or polypeptide. Either qualitative or quantitative methods can be used.
  • the presence of polypeptide products of PAKl polynucleotide can be determined, for example, using a variety of techniques known in the art, including immunochemical methods such as radioimmunoassay, Western blotting, and immunohistochemistry.
  • polypeptide synthesis can be determined in vivo, in a cell culture, or in an in vitro translation system by detecting inco ⁇ oration of labelled amino acids into PAKl .
  • test compounds for use in the screening assays of the invention can be obtained from any suitable source, e.g., conventional compound libraries.
  • the test compounds can also be obtained using any of the numerous approaches in combinatorial library methods known in the art, including: biological libraries; spatially addressable parallel solid phase or solution phase libraries; synthetic library methods requiring deconvolution; the "one-bead one-compound” library method; and synthetic library methods using affinity chromatography selection.
  • the biological library approach is limited to peptide libraries, while the other four approaches are applicable to peptide, non-peptide oligomer or small molecule libraries of compounds [Lam, (1997)]. Examples of methods for the synthesis of molecular libraries can be found in the art. Libraries of compounds may be presented in solution or on beads, bacteria, s ores, plasmids or phage.
  • Computer modeling and searching technologies permit identification of compounds, or the improvement of already identified compounds, that can modulate PAKl expression or activity. Having identified such a compound or composition, the active sites or regions are identified. Such sites might typically be the enzymatic active site, regulator binding sites, or ligand binding sites.
  • the active site can be identified using methods known in the art including, for example, from the amino acid sequences of peptides, from the nucleotide sequences of nucleic acids, or from study of complexes of the relevant compound or composition with its natural ligand. In the latter case, chemical or X-ray crystallographic methods can be used to find the active site by finding where on the factor the complexed ligand is found.
  • the three dimensional geometric structure of the active site is determined. This can be done by known methods, including X-ray crystallography, which can determine a complete molecular structure. On the other hand, solid or liquid phase NMR can be used to determine certain intramolecular distances. Any other experimental method of structure determination can be used to obtain partial or complete geometric structures.
  • the geometric structures may be measured with a complexed ligand, natural or artificial, which may increase the accuracy of the active site structure determined.
  • the methods of computer based numerical modeling can be used to complete the structure or improve its accuracy.
  • Any recognized modeling method may be used, including parameterized models specific to particular biopolymers such as proteins or nucleic acids, molecular dynamics models based on computing molecular motions, statistical mechanics models based on thermal ensembles, or combined models.
  • standard molecular force fields representing the forces between constituent atoms and groups, are necessary, and can be selected from force fields known in physical chemistry.
  • the incomplete or less accurate experimental structures can serve as constraints on the complete and more accurate structures computed by these modeling methods.
  • candidate modulating compounds can be identified by searching databases containing compounds along with information on their molecular structure. Such a search seeks compounds having structures that match the determined active site structure and that interact with the groups defining the active site. Such a search can be manual, but is preferably computer assisted. These compounds found from this search are potential PAKl modulating compounds.
  • these methods can be used to identify improved modulating compounds from an already known modulating compound or ligand.
  • the composition of the known compound can be modified and the structural effects of modification can be determined using the experimental and computer modeling methods described above' applied to the new composition.
  • the altered structure is then compared to the active site structure of .the compound to determine if an improved fit or interaction results. Li this manner systematic variations in composition, such as by varying side groups, can be quickly evaluated to obtain modified modulating compounds or ligands of improved specificity or activity.
  • PAKl is expressed in various human tissues.
  • CNS disorders include disorders of the central nervous system as well as disorders of the peripheral nervous system.
  • CNS disorders include, but are not limited to brain injuries, cerebrovascular diseases and their consequences, Parkinson's disease, corticobasal •degeneration, motor neuron disease, dementia, including ALS, multiple sclerosis, traumatic brain injury, stroke, post-stroke, post-traumatic brain injury, and small-vessel cerebrovascular disease.
  • Dementias such as Alzheimer's disease, vascular dementia, dementia with Lewy bodies,' frontotemporal dementia and Parkinsonism linked to chromosome 17, frontotemporal dementias, including Pick's disease, progressive nuclear palsy, corticobasal degeneration, Huntington's disease, ' thalamic degeneration, Creutzfeld-Jakob dementia, HIV dementia, schizophrenia with dementia, and Korsakoff s psychosis, within the meaning of the definition are also considered to be CNS disorders.
  • CNS disorders such as mild cognitive impairment, age-associated memory impairment, age-related cognitive decline, vascular cognitive impairment, attention deficit disorders, attention deficit hyperactivity disorders, and memory disturbances in children with learning disabilities are also considered to be CNS disorders. Pain, within the meaning of this definition, is also considered to be a CNS disorder. Pain can be associated with CNS disorders, such as multiple sclerosis, spinal cord injury, sciatica, failed back surgery syndrome, traumatic brain injury, epilepsy, Parkinson's disease, post-stroke, and vascular lesions in the brain and spinal cord (e.g., infarct, hemonhage, vascular malformation).
  • CNS disorders such as multiple sclerosis, spinal cord injury, sciatica, failed back surgery syndrome, traumatic brain injury, epilepsy, Parkinson's disease, post-stroke, and vascular lesions in the brain and spinal cord (e.g., infarct, hemonhage, vascular malformation).
  • Non-central neuropathic pain includes that associated with post mastectomy pain, phantom feeling, reflex sympathetic dystrophy (RSD), trigeminal neuralgiaradioculopathy, post-surgical pain, HIV/AIDS related pain, cancer pain, metabolic neuropathies (e.g., diabetic neuropathy, vasculitic neuropathy secondary to connective tissue disease), paraneoplastic polyneuropathy associated, for example, with carcinoma of lung, or leukemia, or lymphoma, or carcinoma of prostate, colon or stomach, trigeminal neuralgia, cranial neuralgias, and post-he ⁇ etic neuralgia. Pain associated with peripheral nerve damage, central pain (i.e.
  • Headache pain for example, migraine with aura, migraine without aura, and other migraine disorders
  • episodic and chronic tension-type headache tension-type like .headache, cluster headache, and chronic paroxysmal hemicrania are also CNS disorders.
  • Visceral pain such as pancreatits, intestinal cystitis, dysmenonhea, irritable Bowel syndrome, Crohn's disease, biliary colic, ureteral colic, myocardial infarction and pain syndromes of the pelvic cavity, e.g., vulvodynia, orchialgia, urethral syndrome and protatodynia are also CNS disorders.
  • a disorder of the nervous system are acute pain, for example postoperative pain, and pain after trauma.
  • the human PAKl is highly expressed in the ' following brain tissues: fetal brain, brain, Alzheimer brain, cerebellum, Alzheimer brain cerebellum, Alzheimer cerebral cortex, occipital lobe, parietal lobe, temporal lobe, substantia nigra, caudatum, cerebral peduncles, nucleus accumbens, hippocampus, thalamus, posterovenfral thalamus, dorsalmedial thalamus, spinal cord, spinal cord (ventral horn), spinal cord (dorsal horn), neuroblastoma SH-SY5Y cells, neuroblastoma IMR32 cells.
  • Heart failure is defined as a pathophysiological state in which an abnormality of cardiac function is responsible for the failure of the heart to pump blood at a rate commensurate with the requirement of the metabolizing tissue. It includes all forms of pumping failures such as high-output and low- output, acute and chronic, right-sided or left-sided, systolic or diastolic, independent of the underlying cause. ⁇ •
  • MI Myocardial infarction
  • Ischemic diseases are conditions in which the coronary flow is restricted resulting in a perfusion which is inadequate to meet the myocardial requirement for oxygen.
  • This group of diseases includes stable angina, unstable angina and asymptomatic ischemia.
  • Anhythmias include all forms of atrial and ventricular tachyanhythmias, atrial tachycardia, atrial flutter, atrial fibrillation, atrio-ventricular reentrant tachycardia, preexitation syndrome, ventricular tachycardia, ventricular flutter, ventricular fibrillation, as well, as bradycardic forms of anhythmias.
  • Hypertensive vascular diseases include primary as well as all kinds of secondary arterial hypertension, renal, endocrine, neurogenic, others.
  • the genes may be used as drug targets for the treatment of hypertension as well as for the prevention of all complications arising from cardiovascular diseases.
  • Peripheral vascular diseases are defined as vascular diseases in which arterial and/or venous flow is reduced resulting in an imbalance between blood supply and tissue oxygen demand. It includes chronic peripheral arterial occlusive disease (PAOD), acute arterial thrombosis and embolism, inflammatory vascular disorders, Raynaud's phenomenon and venous disorders.
  • PAOD peripheral arterial occlusive disease
  • acute arterial thrombosis and embolism inflammatory vascular disorders
  • Raynaud's phenomenon Raynaud's phenomenon
  • Atherosclerosis is a cardiovascular disease in which the vessel wall is remodeled, compromising the lumen of the vessel.
  • the atherosclerotic remodeling process involves accumulation of cells, both smooth muscle cells and monocyte/macrophage inflammatory cells, in the intima of the vessel wall. These cells take up lipid, likely from the circulation, to form a mature atherosclerotic lesion.
  • the formation of these lesions is a chronic process, occuning over decades of an adult human life, the majority of the morbidity associated with atherosclerosis occurs when a lesion ruptures, releasing thrombogenic debris that rapidly occludes the artery.
  • myocardial infarction can ensue, and in the worst case, can result in death.
  • the formation of the atherosclerotic lesion can be considered to occur in five overlapping stages such as migration, lipid accumulation, recruitment of inflammatory cells, proliferation of vascular smooth muscle cells, and extracellular matrix deposition.
  • stages such as migration, lipid accumulation, recruitment of inflammatory cells, proliferation of vascular smooth muscle cells, and extracellular matrix deposition.
  • Each of these processes can be shown to occur in man and in animal models of atherosclerosis, but the relative contribution of each to the pathology and clinical significance of the lesion is unclear.
  • Cardiovascular diseases include but are not limited to disorders of the heart and the vascular system like congestive heart failure, myocardial infarction, ischemic diseases of the heart, all kinds of atrial and ventricular anhythmias, hypertensive vascular diseases, peripheral vascular diseases, and atherosclerosis.
  • the risk to develop atherosclerosis and coronary artery or carotid artery disease (and thus the risk of having a heart attack or stroke) increases with the total cholesterol level increasing.
  • hyperlipidemia abnormally high levels of fats (cholesterol, triglycerides, or both) in the blood, may be caused by family history of hyperlipidemia), obesity, a high-fat diet, lack of exercise, moderate to high alcohol consumption, cigarette smoking, poorly controlled diabetes, and an underactive thyroid gland), hereditary hyperlipidemias (type I hyperlipoproteinemia (familial hyperchylomicronemia), type II hyperlipoproteinemia (familial hypercholesterolemia), type HI hyperlipoproteinemia, type IV hyperlipoproteinemia, or type V hyperlipoproteinemia), hypo- lipoproteinemia, lipidoses (caused by abnormalities in the enzymes that metabolize fats), Gaucher's disease, Niemann-Pick disease, Fabry's disease, Wolman's disease, cerebrotendinous xanthomatosis, sitosterolemia, Refsum's disease, or Tay-Sachs
  • hyperlipidemia abnormally high levels of fats (cholesterol, triglycer
  • Kidney disorders may lead to hypertension or hypotension. Examples for kidney problems possibly leading to hypertension are renal artery stenosis, pyelonephritis, glomerulonephritis, kidney tumors, polycistic kidney disease, injury to the kidney, or radiation therapy affecting the kidney. Excessive urination may lead to hypotension.
  • the human PAKl is highly expressed in the following cardiovascular related tissues: heart atrium (left), heart ventricle (left), aorta, aorta sclerotic, coronary artery smooth muscle primary cells, adrenal gland, liver liver cinhosis. Expression in the above mentioned tissues and in particular the differential expression between diseased tissue aorta sclerotic and healthy tissue demonstrates that the human PAKl or mRNA can be utilized to diagnose of cardiovascular diseases. Additionally the activity of the human PAKl can be modulated to treat cardiovascular diseases.
  • the human PAKl is highly expressed in liver tissues: liver liver cinhosis. Expression in liver tissues demonstrates that the human PAKl or mRNA can be utilized to diagnose of dyslipidemia disorders as an cardiovascular disorder. Additionally the activity of the human PAKl can be modulated to treat - but not limited to - dyslipidemia disorders.
  • the human PAKl is highly expressed in adrenal gland. Expression in adrenal gland tissues demonstrates that the human PAKl or mRNA can be utilized to diagnose of blood pressure disorders as an cardiovascular disorder. Additionally the activity of the human PAKl can be modulated to treat - but not limited to - blood pressure disorders as hypertension or hypotension.
  • Hematological disorders comprise diseases of the blood and all its constituents as well as diseases of organs and tissues involved in the generation or degradation of all the constituents of the blood. They include but are not limited to 1) Anemias, 2) Myeloproliferative Disorders, 3) Hemonhagic Disorders, 4) Leukopenia, 5) Eosinophilic Disorders; 6) Leukemias, 7) Lymphomas, 8) Plasma Cell Dyscrasias, 9) Disorders of the Spleen in the course of hematological disorders. Disorders according to 1) include, but are not limited to anemias due to defective or deficient hem synthesis, deficient erythropoiesis.
  • Disorders according to 2) include, but are not limited to polycythemia vera, rumor-associated erythrocytosis, myelofibrosis, thrombocythemia.
  • Disorders according to 3) include, but are not limited to vasculitis, thrombocytopenia, heparin-induced thrombocytopenia, thrombotic thrombocytopenic pu ⁇ ura, hemolytic-uremic syndrome, hereditary and acquired disorders of platelet function, hereditary coagulation disorders.
  • Disorders according to 4) include, but are not limited to duropenia, lymphocytopenia'.
  • Disorders according to 5) include, but are not limited to hypereosinophilia, idiopathic hypereosinophilic syndrome.
  • Disorders according to 6) include, but are not limited to acute myeloic leukemia, acute lymphoblastic leukemia, chronic myelocytic leukemia, chronic lymphocytic leukemia, myelodysplastic syndrome.
  • Disorders according to 7) include, but are not limited to Hodgkin's disease, non-Hodgkin's lymphoma, Burkitt's lymphoma, mycosis fungoides cutaneous T-cell lymphoma.
  • Disorders according to 8) include, but are not limited to multiple myeloma, macroglobulinemia, heavy chain diseases.
  • iron deficiency anemia In extension of the preceding idiopathic thrombocytopenic pu ⁇ ura, iron deficiency anemia, megaloblastic anemia (vitamin B12 deficiency), aplastic anemia, thalassemia, malignant lymphoma bone marrow invasion, malignant lymphoma skin invasion, hemolytic uremic syndrome, giant platelet disease are considered to be hematological diseases too.
  • the human PAKl is highly expressed in the following tissues of the hematological system: leukocytes (peripheral blood), Jurkat (T-cells), bone manow, bone marrow CD 15+ cells, neutrophils peripheral blood.
  • leukocytes peripheral blood
  • Jurkat T-cells
  • bone manow bone marrow CD 15+ cells
  • neutrophils peripheral blood The expression in the above mentioned tissues demonstrates that the human PAKl or mRNA can be utilized to diagnose- of hematological diseases. Additionally the activity of the human PAKl can be modulated to treat hematological disorders.
  • Gastrointestinal diseases comprise primary or secondary, acute or chronic diseases of the organs of the gastrointestinal tract which may be acquired or inherited, benign or malignant or metaplastic, and which may affect the organs of the gastrointestinal tract or the body as a whole. They comprise but are not limited to 1) disorders of the esophagus like achalasia, vigoruos achalasia, dysphagia, cricopharyngeal incoordination, pre-esophageal dysphagia, diffuse esophageal spasm, globus sensation, Banett's metaplasia, gastroesophageal ' reflux, 2) disorders of the stomach and duodenum like functional dyspepsia, inflammation, of the gastric mucosa, gastritis, stress gastritis, chronic erosive gastritis, atrophy of gastric glands, metaplasia of gastric tissues, gastric ulcers, duodenal ulcers, neoplasms of the stomach, 3) disorders of the pancre
  • Liver diseases comprise primary or secondary, acute or chronic diseases or injury of the liver which may be acquired or inherited, benign or malignant, and which may affect the liver or the body as a whole. They comprise but are not limited to disorders of the bilirubin metabolism, jaundice, syndroms of Gilbert's, Crigler-Najjar, Dubin- Johnson and Rotor; intrahepatic cholestasis, hepatomegaly, portal hypertension, ascites, Budd-Chiari syndrome, portal-systemic encephalopathy, fatty liver, steatosis, Reye's syndrome, liver diseases due to alcohol, alcoholic hepatitis or cinhosis, fibrosis and cinhosis, fibrosis and cinhosis of the liver due to inborn enors of metabolism' or exogenous substances, storage diseases, syndromes of Gaucher's, Zellweger's, Wilson's - disease, acute or chronic hepatitis, viral hepatitis and its variant
  • the human PAKl is highly expressed in the following tissues of the gastroenterological system: stomach tumor, colon, colon tumor, small intestine, ileum, rectum, salivary gland, liver liver cinhosis.
  • the expression in the above mentioned tissues and in particular the differential expression between diseased tissue liver liver cinhosis and healthy tissue liver demonsfrates that the human PAKl or mRNA can be utilized to diagnose of gastroenterological disorders. Additionally the activity of the human PAKl can be modulated to treat gastroenterological disorders.
  • the endocrine system consists of a group of organs whose main function is to produce and secrete hormones directly into the bloodstream.
  • the major organs of the endocrine system are the hypothalamus, the pituitary gland, thyroid gland, the parathyroid glands, the islets of the pancreas, the adrenal glands, the testes, and the ovaries.
  • the pituitary gland coordinates many functions of the other endocrine glands, but some pituitary hormones have direct effects.
  • the insulin-secreting cells of the pancreas respond to glucose and fatty acids.
  • Parathyroid cells respond to calcium and phosphate.
  • the adrenal medulla (part of the adrenal gland) responds to direct stimulation by the parasympathetic nervous system.
  • Diabetes mellitus is a disorder in which blood levels of glucose are abnormally high because the body doesn't release or use insulin adequately. People with type I diabetes mellitus (insulin-dependent diabetes) produce little or no insulin at all. In type I diabetes more than 90 percent of the insulin-producing cells (beta cells) of the pancreas are permanently destroyed. The resulting insulin deficiency is severe, and to survive, a person with type I diabetes must regularly inject insulin.
  • type II diabetes mellitus non-insulin-dependent diabetes
  • the body develops resistance to insulin effects, resulting in a relative insulin deficiency.
  • pancreas has two major functions: to secrete fluid containing digestive enzymes into the duodenum and to secrete the hormones insulin and glucagon.
  • Chronic pancreatitis is a longstanding inflammation of the pancreas.
  • An insulinoma is a rare type of pancreatic tumor that secretes insulin.
  • the symptoms of an insulinoma result from low blood glucose levels.
  • a gasfrinoma is a pancreatic tumor that produces excessive levels of the hormone gastrin, which stimulates the stomach to secrete acid and enzymes, causing peptic ulcers.
  • a glucagonoma is a tumor that produces the hormone glucagon, which raises the level of glucose in the blood and produces a distinctive rash.
  • Diabetes insipidus is a disorder in which insufficient levels of antidiuretic hormone cause excessive thirst (polydipsia) and excessive production of very dilute urine (polyuria). Diabetes insipidus results from the decreased production of antidiuretic hormone (vasopressin).
  • the body has two adrenal glands.
  • the medulla of the adrenal glands secretes hormones such as adrenaline (epinephrine) that affect blood pressure, heart rate, sweating, and other activities also regulated by the sympathetic nervous system.
  • the cortex secretes many different hormones, including corticosteroids (cortisone-like hormones), androgens (male hormones), and mineralocorticoids, which control blood pressure and the levels of salt and potassium in the body.
  • Addison's disease as characterized by underactive adrenal glands is Addison's disease (adrenocortical insufficiency).
  • Adrenal Glands Several disorders are characterized by overactive Adrenal Glands.
  • the causes can be changes in the adrenal glands themselves or overstimulation by the pituitary gland. Examples of these diseases are listed in the following.
  • the thyroid is a small gland located under the Adam's apple. It secretes thyroid hormones, which control the metabolic rate. The thyroid gland traps- iodine arid processes it into thyroid hormones. The euthyroid sick syndrome is characterized by lack of conversion, of the T4 form of thyroid hormone to the T3 form. Hyperthyroidism (overact ⁇ ve thyroid gland, production of too much hormone) may have several causes. Thyroiditis (an inflammation of the thyroid gland), typically leads to a phase of hyperthyroidism. The inflammation may damage the thyroid gland, so that in later stages the disease is characterized by transient or permanent underactivity (hypothyroidism). Toxic thyroid nodules (adenomas) often produce thyroid hormone in large quantities.
  • Toxic multinodular goiter is a disorder in which there are many nodules. Graves' disease (toxic diffuse goiter) is believed to be caused by an antibody that stimulates the thyroid to produce too much thyroid hormone. In toxic nodular goiter, one or more nodules in the thyroid produce too much thyroid hormone and aren't under the control of thyroid-stimulating hormone. Secondary hyperthyroidism may (rarely) be caused by a pituitary tumor that secretes too much thyroid-stimulating hormone, by resistance of the pituitary to thyroid hormone, which results in the pituitary gland secreting too much thyroid-stimulating hormone, or by a hydatidiform mole in women. Thyroid storm is a sudden extreme overactivity of the thyroid gland is a life-threatening emergency requiring prompt treatment.
  • hypothyroidism is a condition in which the thyroid gland is underactive and produces too little thyroid hormone. Very severe hypothyroidism is called myxedema. In Hashimoto's thyroiditis (autoimmune thyroiditis) the thyroid gland is often enlarged, and hypothyroidism results because the gland's functioning areas are gradually destroyed. Rarer causes of hypothyroidism include some inherited disorders which are caused by abnormalities ' of the enzymes in thyroid cells. In other rare disorders, either the hypothalamus or the pituitary gland fails to secrete enough of the hormone needed to stimulate normal thyroid function.
  • Thyroiditis are silent lymphocytic thyroiditis, Hashimoto's thyroiditis, or subacute granulomatous thyroiditis.
  • Thyroid cancer is any one of four main types of malignancy of the thyroid: papillary, follicular, anaplastic, or medullary.
  • the pituitary is a pea-sized gland that sits in a bony structure (sella turcica) at the base of the brain. The sella turcica protects the pituitary but allows..very little room for expansion. If the pituitary enlarges, it tends to push upward, often pressing on the areas of the brain that carry signals from the eyes, possibly resulting in headaches or "impaired vision.
  • the pituitary gland has two distinct parts: the anterior (front) and the posterior (back) lobes.
  • the anterior lobe produces (secretes) hormones that ultimately control the function of the thyroid gland, adrenal glands, and reproductive organs (ovaries and testes); milk production (lactation) in the breasts; and overall body growth. It also produces hormones that cause the skin to darken and that inhibit pain sensations.
  • the posterior lobe produces hormones that regulate water balance, stimulate the let- down of milk from the breasts in lactating women, and stimulate contractions of the uterus.
  • Examples for disorders of the pituitary gland are Empty Sella Syndrome; hypopituitarism (an underactive pituitary gland); acromegaly, which is excessive growth caused by oversecretion of growth hormone, which is almost always caused by a benign pituitary tumor (adenoma); galactonhea, which is the production of breast milk in men or in women who aren't breastfeeding, in both sexes, the most common cause of . galactonhea is a prolactin-producing tumor (prolactinoma) in the pituitary gland.
  • prolactin-producing tumor prolactinoma
  • the human PAKl is highly expressed in the following tissues of the endocrinological system: adrenal gland, thyroid, pancreas, pancreas liver cinhosis.
  • the expression in the above mentioned tissues and in particular the differential expression between diseased tissue pancreas liver cinhosis and healthy tissue pancreas demonstrates that the human PAKl or mRNA can be utilized to diagnose of endocrinological disorders. Additionally the activity of the human PAKl can be modulated to treat endocrinological disorders.
  • the skin serves several functions. It's an multi-layered organ system that builds an effective protective cover and regulates body temperature, senses painful and pleasant stimuli, keeps substances from entering the body, and provides a shield from the sun's harmful effects. Skin color, texture, and folds help mark people as individuals. Thus, skin disorders or diseases often have important consequences for physical and mental health. Skin disorders include, but are not limited to the conditions described in the following.
  • Itching is a sensation that instinctively demands scratching, which may be caused by a skin condition or a systemic diseas.
  • Superficial Skin Disorders affect the uppermost layer of the skin, the stratum corneum or the keratin layer, and it consists of many layers of flattened, dead cells and acts as a barrier to protect the underlying tissue from injury and infection. Disorders of the superficial skin layers involve the stratum corneum and deeper layers of the epidermis.
  • ichthyosis Dry skin often occurs in people past middle .age, severe dry skin (ichthyosis) results from an inherited scaling disease, such as ichthyosis vulgaris or epidermolytic hyperkeratosis. Ichthyosis also results from nonhereditary disorders, such as leprosy, underactive thyroid, lymphoma, ADDS, and sarcoidosis.
  • Keratosis pilaris is a common disorder in which dead cells shed from the upper layer of skin and form plugs that fill the openings of hair follicles.
  • a callus is an area on the stratum corneum or keratin layer, that becomes abnormally thick in response to repeated rubbing.
  • a corn is a pea-sized, thickened area of keratin that occurs on the feet.
  • Psoriasis is a chronic, recuning disease recognizable by silvery scaling bumps and various-sized plaques (raised patches). An abnormally high rate of growth and turnover of skin cells causes the scaling.
  • Pityriasis rosea is a mild disease that causes scaly, rose-colored, inflamed skin. Pityriasis rosea is possibly caused by an infectious agent, although none has been identified.
  • Lichen planus a recuning itchy disease, starts as a rash of small discrete bumps that then combine and become rough, scaly plaques (raised patches).
  • Dermatitis is an inflammation of the upper layers of the skin, causing blisters, redness, swelling, oozing, scabbing, scaling, and usually itching:
  • dermatitis are contact dermatitis, or chronic dermatitis of the hands and feet, e.g. Pompholyx.
  • dermatitic disorders are atopic dermatitis, sebonheic dermatitis, nummular dermatitis, generalized exfoliative de ⁇ natitis, stasis dermatitis, or localized scratch de ⁇ natitis (lichen simplex chronicus, neurodermatitis).
  • Other skin disorders are caused by inflammation. The skin can break out in a variety of rashes, sores, and blisters. Some skin eruptions can even be life threatening.
  • Drug rashes are side effects of medications, mainly allergic reactions to medications.
  • Toxic epidermal necrolysis is a life-threatening skin 'disease in which the top layer of the skin peels off in sheets. This condition can be caused by a reaction to a drug, or by some other serious disease.
  • Erythema multiforme, o ften caused by he ⁇ es simplex is a disorder characterized by patches of red, raised skin that often look like targets and usually are distributed symmetrically over the body.
  • Erythema nodosum is an inflammatory disorder that produces tender red bumps (nodules) under the skin, most often over the shins but occasionally on the arms and other areas.
  • Granuloma annulare is a chronic skin condition of unknown cause in which small, firm, raised bumps form a ring with normal or slightly sunken skin in the center.
  • Some skin disorders are characterized as blistering diseases.
  • Pemphigus is an uncoi ⁇ mon, sometimes fatal, disease in which blisters (bullae) of varying sizes break out on the skin, the lining of the mouth, and other mucous membranes.
  • Bullous pemphigoid is an autoimmune disease that causes blistering.
  • Dermatitis he ⁇ etiformis is an autoimmune disease in which clusters of intensely itchy, small blisters and hive-like spellings break out and persist.
  • proteins in wheat, rye, barley, and oat products activate the immunfe system, which attacks parts of the skin and somehow causes the rash and itching.
  • Sweating disorders also belong to skin disorders.
  • Prickly heat is an itchy skin rash caused by trapped sweat.
  • Excessive sweating may affect the entire surface of the skin, but often it's limited to the palms, soles, armpits, or groin.
  • the affected area is often pink or bluish white, and in severe cases the skin may be cracked, scaly, and soft, especially on the feet.
  • Skin disorders can affect the sebaceous glands.
  • Acne is a common skin condition in which the skin pores become clogged, leading to pimples and inflamed, infected abscesses (collections of pus). Acne tends to develop in teenagers.
  • Rosacea is a persistent skin disorder that produces redness, tiny pimples, and broken blood vessels, usually on the central area of the face.
  • Perioral dermatitis is a red, often bumpy rash around the mouth and on the chin.
  • a sebaceous cyst (keratinous cyst) is a slow-growing bump containing dead skin, skin excretions, and other skin particles. These cysts may be small and can appear anywhere.
  • Hair disorders also are skin disorders. Hair disorders include excessive hairiness, baldness, and ingrown beard hairs.
  • the skin can be infected by bacteria.
  • Bacterial skin infections can range in seriousness from minor acne to a life-threatening condition, such as staphylococcal scalded skin syndrome.
  • the most common bacterial skin infections are caused by Staphylococcus and Streptococcus.
  • Risk factors for skin infections are for example diabetes, AIDS or skin leasons.
  • Impetigo is a skin infection, caused by Staphylococcus or Streptococcus, leading to the formation of small pus-filled blisters (pustules).
  • Folliculitis is an inflammation of the hair follicles caused by infection with Staphylococcus. The infection damages the hairs, which can be easily pulled out.
  • Boils are large, tender, swollen, raised areas caused by staphylococcal infection around hair follicles.
  • Carbuncles are clusters of boils that result in extensive sloughing of skin and scar formation. Carbuncles develop and heal more slowly than single boils and may lead to fever and fatigue-
  • Erysipelas is a skin infection caused by Streptococcus. A shiny, red, slightly swollen, tender rash develops, often with small blisters. Lymph nodes around the infected area may become enlarged and painful. Cellulitis is a spreading infection in, and sometimes beneath, the deep layers of the skin. Cellulitis most often results from a streptococcal infection or a staphylococcal infection. However, many other bacteria can also cause cellulitis.
  • Paronychia is an infection around the edge of a fingernail or toenail. Paronychia can be caused by many different bactena, including Pseudomonas and Proteus, and by fungi, such as Candida.
  • Staphylococcal scalded skin syndrome is a widespread skin infection that can lead to toxic shock syndrome, in which the skin peels off as though burned. Certain types of staphylococci produce a toxic substance that causes the top layer of skin (epidermis) to split from the rest of the skin.
  • Erythrasma is an infection of the top layers of the skin by the bacterium Corynebacterium minutissimum.
  • Skin infections are often caused by fungi. Fungi that infect the skin (dermatophytes) live only in the dead, topmost layer (stratum corneum) and don't penetrate deeper. Some fungal infections cause no symptoms or produce only a small amount of initation, scaling, and redness. Other fungal infections cause itching, swelling, blisters, and severe scaling.
  • Ringworm is a fungal skin infection caused by several different fungi and generally classified by its location on the body.
  • Athlete's foot (foot ringworm, caused by eit er Trichophyton or Epidermophyton), jock itch (groin ringworm, can be caused by a variety of fungi and yeasts), scalp ringworm, caused by Trichophyton or Microsporum), nail ringworm and body -ringworm (caused by Trichophyton).
  • Candida infection is an infection by the yeast Candida.
  • Candida usually infects the skin and mucous membranes, such as the lining of the mouth and vagina. Rarely, it invades deeper tissues as well as the blood, causing life-threatening systemic candidiasis.
  • the following types of Candida infections can be distinguished: Infections in skinfolds (intertriginous infections), vaginal and penile Candida infections (vulvovaginitis), thrush, Perleche (candida infection at the comers of the mouth), candidal paronychia (candida growing in the nail beds, produces painful swelling and pus).
  • Tinea versicolor is a fungal infection that causes white to liglht brown patches on the skin.
  • the skin can also be affected by parasites, mainly tiny insects or worms.
  • Scabies is a mite infestation that produces tiny reddish pimples and severe itching. Scabies is caused by the itch mite Sarcoptes scabiei. Lice infestation (pediculosis) causes intense itc ing and can affect almost any area of the skin. Head lice and pubic lice are two different species-
  • Creeping eruption (cutaneous larva migrans) is a hookworm infection transmitted from warm, moist soil to exposed skin. The infection is caused by a hookworm that normally inhabits dogs and cats.
  • Sunlight can cause severe skin damage. Sunburn results from an overexposure to ultraviolet B (UNB) rays. Some sunburned people develop a fever, chills, and weakness, and those with very bad sunburns even may go into shock-low blood pressure, and fainting.
  • UV B ultraviolet B
  • Drugs can cause skin photos ensitivity reactions which can occur after only a few minutes of sun exposure. These reactions include redness, peeling, hives, blisters, and thickened, scaling patches (photosensitivity). ,
  • Pigment -Disorders Some skin disorders are characterized as Pigment -Disorders.
  • Albinism is a rare, inherited disorder in which no melanin is formed.
  • Vitiligo is a condition in which a loss of melanocytes results in smooth, whitish patches of skin, which may occur after unusual physical trauma and tends to occur with certain other diseases, including Addison's disease, diabetes, pernicious anemia, and thyroid disease.
  • Tinea versicolor is a fungal infection of the skin that sometimes results in hype ⁇ igmentation.
  • Melasma appears on the face (usually the fore ead, cheeks, temples, and jaws) as a roughly symmetric group of dark brown patches of pigmentation that are often clearly delineated. Skin growths, which are abnormal accumulations of different types of cells, may be present at birth or develop later. Noncancerous (benign) growth and cancerous (malignartt) growth types are distinguished.
  • Moles are small, usually dark, skin growths that develop from pigment-producing cells in the skin (melanocytes). Most moles are harmless. However, noncancerous moles can develop into malignant melanoma.
  • Skin tags are soft, small, flesh-colored or slightly darker skin flaps that appear mostly on the neck, in the armpits, or in the groin.
  • Lipomas are soft deposits of fatty material that grow under the skin, causing round or oval lumps.
  • Angiomas are collections of abnormally dense blood or lymph vessels that acre usually located in and below the skin and that cause red or pu ⁇ le discolorations.
  • angiomas examples include port-wine stains, strawberry marks, cavernous emangiomas, spider angiomas, and lymphangiomas.
  • Pyogenic granulomas are scarlet, brown, or blue-black slightly raised areas caused by increased growth of capillaries (the smallest blood vessels) and swelling of the sunounding tissue.
  • Sebonheic keratoses (sometimes called sebonheic warts) are flesh-cplore-d, brown, or black growths that can appear anywhere on the skin.
  • Dermatofibromas are small, red-to-brown bumps (nodules) that result from an accumulation of fibroblasts, the cells that populate the soft tissue under the skin.
  • Keratoacanthomas are round, firm, usually flesh-colored growths that have an unusual central crater containing a pasty material.
  • Keloids are smooth, shiny, slightly pink, often dome-shaped, proliferative growths of fibrous tissue that form over areas of injury or over surgical wounds.
  • Skin cancer is the most common form of cancer, but most types of skin cancers are curable.
  • Basal cell carcinoma is a cancer that originates in the lowest layer of the epidermis.
  • Squamous cell carcinoma is cancer that originates in the middle layer of the epidermis.
  • Bowen's disease is a form of squamous cell carcinoma that's confined to the epidermis and hasn't yet invaded the underlying dermis.
  • Melanoma is a cancer that originates in the pigment-producing cells of the skin (melano ⁇ ytes).
  • Kaposi's sarcoma is a cancer that originates in the blood vessels, usually of the skin.
  • Paget's disease is a rare type of skin cancer that looks like an inflamed, reddened patch of skin (dermatitis); it originates in glands in or under the skin.
  • the human PAKl is highly expressed in the following dermatological tissues: skin.
  • the expression in the above mentioned tissues demonstrates that the human PAKl or mRNA can be utilized to diagnose of dermatological diseases. Additionally the activity of the human PAKL1 can be modulated to treat those diseases.
  • Components of the musculoskeletal system are skeleton, muscles, tendons, ligaments, and other components of joints. Disorders of the musculoskeletal system often cause chronic pain and physical disability. They range from injures, infections, inflammation or other types of disorders. Examples of musculoskeletal disorders are presented in the following.
  • osteoporosis examples are osteoporosis, postmenopausal • osteoporosis, senile osteoporosis, secondary osteoporosis, idiopathic juvenile osteoporosis, Paget's disease of the bone, osteocb-ondromas (osteocartilaginous exostoses), tumors of the bone (benign chondromas, chondroblastomas, chondromyxoid fibromas, osteoid osteomas, giant cell tumors of the bone, multiple --myeloma, osteosarcoma (osteogenic sarcoma), fibrosarcomas and malignant fibrous histiocytomas, chondrosarcomas, Ewing's tumor (Ewing's sarcoma), malignant lymphoma of bone (reticulum cell sarcoma, metastatic tumors of the bone), osteoarthritis, and gout and Pseudogout.
  • osteocb-ondromas osteo
  • disorders of joints and connective tissue are rheumatoid arthritis, psoriatic arthritis, discoid lupus erythematosus, systemic lupus erythematosus, scleroderma (systemic sclerosis), Sj ⁇ gren's syndrome, connective tissue disease, polymyositis and dermatomyositis, relapsing polychondritis, vasculitis, polyarteritis nodosa, polymyalgia rheumatica, temporal arteritis, Wegener's granulomatosis, Reiter's syndrome, Behcet's syndrome, ankylosing sponcdylitis, or Charcot's joints (neuropathic joint disease).
  • osteomyelitis examples are osteomyelitis, and infectious arthritis.
  • disorders of muscles, bursas, and tendons are spasmodic torticollis, f ⁇ bromyalgia syndromes (myofascial pain syndromes, fibromyositis), bursitis, tendinitis and tenosynovitis.
  • Foot problems are, for example ankle sprain, foot fractures, heel spurs, Sever's disease, posterior achilles tendon bursitis, anterior achilles tendon bursitis, posterior tibial neuralgia, pain Ln the ball of the foot (caused by damage to the nerves between the toes or to the joints between the toes and foot), onychomycosis, or nail discoloration.
  • the human PAKl is highly expressed in the. following muscle/skeleton tissues: cartilage.
  • the expression in muscle/skleleton tissues demonstrates that the human PAKl or mRN-A can be utilized to diagnose of diseases of the muscle/skeleton system. Additionally the activity of the human PAKl can be modulated to treat those diseases.
  • Cancer disorders within the scope of this definition comprise any disease of an organ or tissue in mammals characterized by poorly controlled or uncontrolled multiplication of normal or abnormal cells in that tissue and its effect on the body as a whole.
  • Cancer diseases within the scope of the definition comprise benign neoplasms, dysplasias, hype ⁇ lasias as well as neoplasms showing metastatic growth or any other transformations like e.g. leukoplakias which often precede a breakout of cancer.
  • Cells and tissues are cancerous when they grow more rapidly tham normal cells, displacing or spreading into the sunounding healthy tissue or any other tissues of the body described as metastatic growth, assume abnormal shapes and sizes, show changes in their nucleocytoplasmatic ratio, nuclear polychro-nasia, and finally may cease.
  • Cancerous cells and tissues may affect the body as a whole when causing paraneoplastic syndromes or if cane er occurs within a vital organ or tissue, normal function will be impaired or halted, with possible fatal results.
  • the ultimate involvement of a vital organ by cancer, either primary or metastatic, may lead to the death of the mammal affected. Cancer tends to spread, and the extent of its spread is usually related to an individual's chances of surviving the disease.
  • Cancers are generally said to " be in one of three stages of growth: early, or localized, when a tumor is still confined to the tissue of origin, or primary site; direct extension, where cancer cells from the tumour have invaded adjacent tissue or have spread only to regional lymph nodes; or metastasis, in which cancer cells have migrated to distant parts of the body from the primary site, via the blood or lymph systems, and have established secondary sites of infection.
  • Cancer is said to be malignant because of its tendency to cause death if not treated. Benign tumors usually do not cause death, although they ma-y if they interfere with a normal body function by virtue of their location, size, or paraneoplastic side effects. Hence benign tumors fall under the definition of cancer within the scope of this clefinition as well.
  • cancer cells divide at a higher rate than do normal cells, but the distinction between the growth of cancerous and normal tissues is not so much the rapidity of cell division in the former as it is the partial or complete loss of growth restraint in cancer cells and their failure to differentiate into a useful, limited tissue of the type that characterizes the functional equilibrium of growth of normal tissue.
  • Cancer tissues may express certain molecular receptors and probably are influenced by the host's susceptibility and immunity and it is known that certain cancers of the breast and prostate, for example, are considered dependent on specific hormones for their existence.
  • cancer under the scope of the definition is not limited to simple benign neoplasia but comprises any other benign and-malign neoplasia like 1) Carcinoma, 2) Sarcoma, 3) Carcinosarcoma, 4) Cancers of the blood-forming tissues, 5) tumors of nerve tissues including the brain, 6) cancer of skin cells. Cancer according to 1) occurs in epithelial tissues, which cover the outer body (the skin) and line mucous membranes and the inner cavitary structures of organs e.g. such as the breast, lung, the respiratory and gastrointestinal tracts, the endocrine glands, and the genitourinary system.
  • Ductal or glandular elements may persist in epithelial tumors, as in adenocarcinomas like e.g. thyroid adenocarcinoma, gastric adenocarcinoma, uterine adeno- carcinoma.
  • adenocarcinomas like e.g. thyroid adenocarcinoma, gastric adenocarcinoma, uterine adeno- carcinoma.
  • Cancer according to 2) develops in connective tissues, including fibrous tissues, adipose (fat) tissues, muscle, blood vessels, bone, and cartilage like e.g. osteogenic sarcoma; liposarcoma, fibrosarcoma, synovial sarcoma.
  • Cancer according to 3) is cancer that develops in both epithelial and connective tissue.
  • Cancer disease within the scope of this definition may be primary or secondary, whereby primary indicates that the cancer originated in the tissue where it is found rather than was established as a secondary site through metastasis from another lesion.
  • Cancers and tumor diseases within the scope of this definition may be benign or malign and may affect all anatomical structures of the body of a mammal.
  • cancers and tumor diseases of I) the bone manow and bone manow derived cells (leukemias), II) the endocrine and exocrine glands like e.g. thyroid, parathyroid, pituitary, adrenal glands, salivary glands, pancreas m) the breast, like e.g.
  • malignant osteogenic sarcoma benign osteoma, cartilage tumors; like malignant chondrosarcoma or benign chondroma; bone manow tumors like malignant myeloma or benign eosinophilic granuloma, as well as metastatic tumors from bone tissues at other locations of the body;
  • X) the mouth, throat, larynx, and the esophagus XI) the urinary bladder and the internal and exte al organs and structures of the urogenital system of male and female like ovaries, uterus, cervix of the uterus, testes, and prostate gland, XII) the prostate, XDI) the pancreas, like ductal carcinoma of the pancreas;
  • XTV the lymphatic tissue like lymphomas and other tumors of lymphoid origin, XV) the skin, XVI) cancers and tumor diseases of all anatomical structures belonging to the respiration and respiratory systems including thora
  • the human PAKl is highly expressed in the following cancer tissues: stomach tumor, colon tumor, Jurkat (T-cells), ovary tumor, prostate tumor.
  • the expression in the above mentioned tissues and in particular the differential expression between diseased tissue stomach tumor and healthy tissue stomach, between diseased tissue colon tumoi 1 and healthy tissue colon, between diseased tissue Jurkat (T-cells) and healthy tissue , between diseased tissue ovary tumor and healthy tissue ovary, between diseased tissue prostate tumor and healthy tissue prostate demonsfrates that the human PAKl or mRNA can be utilized to diagnose of cancer. Additionally the activity of the human PAKl can be modulated to treat cancer.
  • feflammatory diseases comprise diseases triggered by cellular or non-cellular mediators of the immune system or tissues causing the inflammation of body tissues and subsequently producing an acute or chronic inflammatory condition.
  • inflammatory diseases are hypersensitivity reactions of type I - IV, for example but not limited to hypersensitivity diseases of the lung including asthma, atopic diseases, allergic rhinitis or conjunctivitis, angioedema of the lids, hereditary angioedema, antireceptor hypersensitivity reactions and autoimmune diseases, Hashimoto's thyroiditis, systemic lupus erythematosus, Goodpasture's syndrome, pemphigus, myasthenia gravis, Grave's and Raynaud's disease, type B insulin-resistant diabetes, rheumatoid arthritis, psoriasis, Crohn's disease, scleroderma, mixed connective tissue disease, polymyositis, sarcoidosis, glomerulonephritis, acute or chronic host
  • the human PAKl is highly expressed in the following tissues of the immune system and tissues responsive to components of the immune system as well as in the following tissues responsive to mediators of inflammation: pancreas liver cirrhosis, liver liver cinhosis, leukocytes (peripheral blood), bone manow, bone manow CD 15+ cells, neutrophils peripheral blood.
  • pancreas liver cirrhosis pancreas liver cirrhosis
  • liver liver cinhosis leukocytes (peripheral blood)
  • bone manow bone manow CD 15+ cells
  • neutrophils peripheral blood neutrophils peripheral blood.
  • the expression in the above mentioned tissues and in particular the differential expression between diseased tissue pancreas liver cinhosis and healthy tissue pancreas, between diseased tissue liver liver cinhosis and healthy tissue liver demonstrates that the human PAKl or mRNA can be utilized to diagnose of inflammatory diseases. Additionally the activity of the human PAKl can be modulated to treat inflammatory diseases. ' • .
  • Asthma is thought to arise as a result of interactions between multiple genetic and environmental factors and is characterized by three major features: 1) intermittent and reversible airway obstruction caused by bronchoconstriction, increased mucus production, and thickening of the walls of the airways that leads to a nanowing of the airways, 2) airway hypenesponsiveness, and 3) airway inflammation.
  • Certain cells are critical to the inflammatory reaction of asthma and they include T cells and antigen presenting cells, B cells that produce IgE, and mast cells, basophils, eosinophils, and other cells that bind IgE. These effector cells accumulate at the site of allergic reaction in the airways and release toxic products that contribute to the acute pathology and eventually to tissue destruction related to the disorder.
  • Other resident cells such as smooth muscle cells, lung epithelial cells, mucus-producing cells, and nerve cells may also be abnormal in individuals with asthma and may contribute to its pathology.
  • airway obstruction of asthma presenting clinically as an intermittent wheeze and shortness of breath, is generally the most pressing symptom of the disease requiring immediate treatment, the inflammation and tissue destruction associated with the disease can lead to ineversible changes that eventually make asthma a chronic and disabling disorder requiring long-term management.
  • COPD chronic obstructive pulmonary (or airways) disease
  • COPD chronic obstructive pulmonary (or airways) disease
  • Emphysema is characterised by destruction of alveolar walls leading to abnormal enlargement of the air spaces of the lung.
  • Chronic bronchitis is defined clinically as the presence of chronic productive cough for three months in each of two successive years.
  • airflow obstruction is usually progressive and is only partially reversible. By far the most important risk factor for development of COPD is cigarette smoking, although the disease does also occur in non-smokers.
  • the human PAKl is highly expressed in the following tissues of the respiratory system: leukocytes (peripheral blood), bone manow CD15+ cells, devisrophils peripheral blood, fetal lung, lung right upper lobe, lung right mid lobe, lung right lower lobe, trachea, primary bronchia, bronchial epithelial cells.
  • leukocytes peripheral blood
  • bone manow CD15+ cells fetal lung
  • fetal lung fetal lung
  • lung right upper lobe lung right mid lobe
  • lung right lower lobe trachea
  • primary bronchia bronchial epithelial cells.
  • the expression in the above mentioned tissues demonstrates that the human PAKl or mRNA can be utilized to diagnose of respiratory diseases. Additionally the activity of the human PAKl can be modulated to treat those diseases. ' Disorders Related to Urology
  • Genitourinary disorders comprise benign and malign disorders of the organs constituting the genitourinary system of female and male, renal diseases like acute or chronic renal failure, immunologically mediated renal diseases like renal transplant rejection, lupus nephritis, immune complex renal diseases, glomerulopathies, nephritis, toxic nephropathy, obstructive uropathies like benign prostatic hype ⁇ lasia (BPH), neurogenic bladder syndrome, urinary incontinence like urge-, stress-, or overflow incontinence, pelvic pain, and erectile dysfunction.
  • renal diseases like acute or chronic renal failure
  • immunologically mediated renal diseases like renal transplant rejection, lupus nephritis, immune complex renal diseases, glomerulopathies, nephritis, toxic nephropathy, obstructive uropathies like benign prostatic hype ⁇ lasia (BPH)
  • neurogenic bladder syndrome urinary incontinence like urge-, stress-, or overflow in
  • the human PAKl is highly expressed in the following urological tissues: spinal cord, spinal cord (ventral horn), spinal cord (dorsal horn), prostate, prostate BPH, prostate tumor, bladder, penis.
  • the expression in the above mentioned tissues- and in particular the differential expression between diseased tissue prostate BPH and healthy tissue prostate demonstrates that the human PAKl or mRNA can be utilized to diagnose of urological disorders. Additionally the activity of the human PAKl can be modulated to treat urological disorders.
  • the human PAKl is highly expressed in spinal cord tissues: spinal cord, spinal cord (ventral hom), spinal cord (dorsal hom). Expression in spinal cord tissues demonstrates that the human PAKl or mRNA can be utilized to diagnose of incontinence 'as an urological disorder.
  • the spinal cord tissues are involved in the neuronal regulation of the urological system. Additionally the activity of the human PAKl can be modulated to treat - but not limited to - incontinence.
  • Metabolic diseases are defined as conditions which result from an abnormality in any of the chemical or biochemical transformations and- their regulating systems essential to producing energy, to regenerating cellular constituents, to eliminating unneeded products arising from these processes, and to regulate and maintain homeostasis in a mammal regardless of whether acquired or the result of a genetic transformation.
  • a single defective fransformation or disturbance of its regulation may produce consequences that are nanow, involving a single body function, or broad, affecting many organs, organ-systems or the body as a whole.
  • Metabolic diseases often are caused by single defects in particular biochemical pathways, defects that are due to the deficient activity of individual •enzymes or molecular receptors leading to the regulation of such enzymes. Hence in a broader sense, disturbances of the underlying genes, their products and their regulation lie well within the scope of this definition of a metabolic disease.
  • metabolic diseas.es may affect 1) biochemical processes and tissues ubiquitous all over the body, 2) the bone, 3) the nervous system, 4) the endocrine system, 5) the muscle including the heart, 6) the skin and nervous tissue, 7) the urogenital system, 8) the homeostasis of body systems like water and electrolytes.
  • metabolic diseases according to 1) comprise obesity, amyloidosis, disturbances of the amino acid metabolism like branched chain disease, hyperaminoacidemia, hyperaminoaciduria, disturbances of the metabolism of urea, hyperammonemia, mucopolysaccharidoses e.g.
  • Maroteaux-Lamy syndrom storage diseases like glycogen storage diseases and lipid storage diseases, glycogenosis diseases like Cori's disease, malabso ⁇ tion diseases like intestinal carbohydrate malabso ⁇ tion, oligosaccharidase deficiency like maltase-, lactase-; sucrase-insufficiency, disorders of the metabolism of fructose, disorders of the metabolism of galactose, galactosaemia, disturbances of carbohydrate utilization like diabetes, hypo ' glycemia, ' disturbances of pyruvate metabolism, hypolipidemia, hypolipoproteinemia, hyperlipidemia, hyperlipoproteinemia, carnitine or camitine acyltransferase deficiency, disturbances of the po ⁇ hyrin metabolism, po ⁇ hyrias, disturbances of the purine metabolism, lysosomal diseases, metabolic diseases of nerves and nervous systems like gangliosidoses, sphingolipidoses, sul
  • metabolic diseases according to 2) comprise osteoporosis, osteomalacia like osteoporosis, osteopenia, osteogenesis imperfecta, osteopefrosis, osteonecrosis, Paget's disease of bone, hypophosphatemia.
  • metabolic diseases according to 3) comprise cerebellar dysfunction, disturbances of brain metabolism like dementia, Alzheimer's disease, Huntington's chorea, Parkinson's disease, Pick's disease, toxic encepha- lopathy, demyelinating neuropathies like inflammatory neuropathy, Guillain-Ba ⁇ e syndrome.
  • metabolic diseases comprise primary and secondary metabolic disorders associated with hormonal defects like any disorder stemming from either an hyperfunction or hypofunction of some hormone-secreting endocrine gland and any combination thereof. They comprise Sipple's syndrome, pituitary gland dysfunction and its effects on other endocrine glands, such as the thyroid, adrenals, ovaries, and testes, acromegaly, hyper- and hypothyroidism, euthyroid goiter, euthyroid sick syndrome, thyroiditis, and thyroid cancer, over- or unde ⁇ roduction of the adrenal steroid hormones, adrenogenital syndrome, Cushing's syndrome, Addison's disease of the adrenal cortex, Addison's pernicious anemia, primary and secondary aldosteronism, diabetes insipidus, carcinoid syndrome, disturbances caused by the dysfunction of the parathyroid glands, pancreatic islet cell dysfunction, diabetes, disturbances of the endocrine syste of the female like
  • metabolic diseases comprise muscle weakness, myotonia, Duchenne's and other muscular dystrophies, dystrophia myotonica of Steinert, mitochondrial myopathies like disturbances of the catabolic metabolism in the muscle, carbohydrate and lipid storage myopathies, glycogenoses, myoglobinuria, malignant hyperthermia, polymyalgia rheumatica, dermatomyositis, primary myocardial disease, cardiomyopathy.
  • metabolic diseases according to 5 comprise muscle weakness, myotonia, Duchenne's and other muscular dystrophies, dystrophia myotonica of Steinert, mitochondrial myopathies like disturbances of the catabolic metabolism in the muscle, carbohydrate and lipid storage myopathies, glycogenoses, myoglobinuria, malignant hyperthermia, polymyalgia rheumatica, dermatomyositis, primary myocardial disease, cardiomyopathy.
  • metabolic diseases according to 6 comprise disorders of the ectoderm, neurofibromatosis, scleroderma and polyarteritis, Louis-Bar syndrome, von Hippel-Lindau disease, Sturge- eber syndrome, tuberous sclerosis, amyloidosis, po ⁇ hyria.
  • metabolic diseases according to 7 comprise sexual dysfunction of the male and female.
  • metabolic diseases according to 8) comprise confused states and seizures due to inappropriate secretion of antidiuretic hormone from the pituitary gland, Liddle's syndrome, Bartter's syndrome, Fanconi's syndrome, renal electrolyte wasting, diabetes insipidus.
  • the human PAKl is highly expressed in the following metabolic disease related tissues: thyroid, pancreas, pancreas liver cinhosis, liver liver cinhosis, cartilage.
  • the expression in the above mentioned tissues and in particular the differential expression between diseased tissue pancreas liver cinhosis and healthy tissue pancreas, between diseased tissue liver liver cinhosis and healthy tissue liver demonsfrates that the human PAKl or mRNA can be utilized to diagnose of metabolic diseases. Additionally the activity of the human PAKl can be modulated to treat metabolic diseases.
  • the present invention provides for both prophylactic and therapeutic methods for cardiovascular diseases, dermatological diseaes, endocrinological diseases, metabolic diseases, inflammation, gastroenterological diseases, cancer, hematological diseases, respiratory diseases, muscle-skeleton diseases, neurological diseases and urological diseases.
  • the regulatory method of the invention involves contacting a cell with an agent that modulates one or more of the activities of PAKl.
  • An agent that modulates activity can be an agent as described herein, such as a nucleic acid or a protein, a naturally-occuning cognate ligand of the polypeptide, a peptide, a peptidomimetic, or any small molecule.
  • the agent stimulates one or more of the biological activities of PAKl. Examples of such stimulatory agents include the active PAKl and nucleic acid molecules encoding a portion of PAKl.
  • the agent inhibits one or more of the biological activities of PAKl. Examples of such inhibitory agents include antisense nucleic acid molecules and antibodies.
  • the present invention provides methods of treating an individual afflicted with a disease or disorder characterized by unwanted expression or activity of PAKl or a protein in the PAKl signaling pathway.
  • the method involves administering an agent like any agent identified or being identifiable by a screening assay as described herein, or combination of such agents that modulate say upregulate or downregulate the expression or activity of PAKl or of any protem in the PAKl signaling pathway.
  • the method involves administering a regulator of PAKl as therapy to compensate for reduced or undesirably low expression or activity of PAKl or a protein in the PAKl signaling pathway.
  • Stimulation of activity or expression of PAKl is desirable in situations in which enzymatic activity or expression is abnormally low and in which increased activity is likely to have a beneficial effect. Conversely, inhibition of enzymatic activity or expression of PAKl is desirable in situations in which activity or expression of PAKl is abnormally high and in which decreasing its activity is likely to have a beneficial effect.
  • This invention further pertains to novel agents ' identified by the above-described screening assays and uses thereof for treatments as described herein.
  • nucleic acid molecules, polypeptides, and antibodies (also refened to herein as "active compounds") of the invention can be inco ⁇ orate.d into pharmaceutical compositions suitable for administration.
  • Such compositions typically comprise the nucleic acid molecule, protein, or antibody and a pharmaceutically acceptable canier.
  • pharmaceutically acceptable carrier is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic ' and abso ⁇ tion delaying agents, and the like, compatible with pharmaceutical administration.
  • the use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated. Supplementary active compounds can also be inco ⁇ orated into the compositions.
  • the invention includes pharmaceutical compositions comprising a regulator of PAKl expression or activity (and/or a regulator of the activity or expression of a protein in the PAKl signaling pathway) as well as methods for preparing such compositions by combining one or more such regulators and a pharmaceutically acceptable canier.
  • pharmaceutical compositions comprising a regulator identified sing the screening assays of the invention packaged with instructions for use.
  • the instructions would specify use of the pharmaceutical composition for treatment of cardiovascular diseases, dermatological diseaes, endocrinological diseases, metabolic diseases, inflammation, gastroenterological diseases, cancer, hematological diseases, respiratory diseases, muscle-skeleton diseases, neurological diseases and urological diseases.
  • the instructions would specify use of the pharmaceutical composition for treatment of cardiovascular diseases, dermatological diseaes, endocrinological diseases, metabolic diseases, inflammation, gastroenterological diseases, cancer, hematological diseases, respiratory diseases, muscle-skeleton diseases, neurological diseases and urological diseases.
  • An inhibitor of PAKl may be produced using methods which are generally known in the art.
  • purified PAKl may be used underneath produce antibodies or to screen libraries of pharmaceutical agents to identify those which specifically bind PAKl.
  • Antibodies to PAKl may also be generated using methods that are well known in the art.
  • Such antibodies may include, but are not limited to, polyclonal, monoclonal, chimeric, single chain antibodies, Fab fragments, and fragments produced by a Fab expression library. Neutralizing antibodies like those which inhibit dimer formation are especially prefened for therapeutic use.
  • the polynucleotides encoding PAKl, or any fragment or complement thereof may be used for therapeutic pu ⁇ oses.
  • the complement of the polynucleotide encoding PAKl may be used in situations in which it would be desirable to block the transcription of the mRNA.
  • cells may be transformed with sequences complementary to polynucleotides encoding PAKl.
  • complementary molecules or fragments may be used to modulate PAKl activity, or to achieve regulation of gene function.
  • sense or antisense oligonucleotides or larger fragments can be designed from various locations along the coding or control regions of sequences encoding PAKl.
  • Expression vectors derived from retroviruses, adenovimses, or he ⁇ es or vaccinia viruses, or from various bacterial plasmids, may be used for delivery of nucleotide sequences to the targeted organ, tissue, or cell population. Methods which are well known to those skilled in the art can be used to construct vectors which will express nucleic acid sequence complementary to the polynucleotides of the gene encoding PAKl. These techniques are described, for example, in [Scott and Smith (1990)].
  • any of the therapeutic methods described above may be applied to any subject in need of such therapy, including, for example, mammals such as dogs, cats, cows, horses, rabbits, monkeys, and most preferably, humans.
  • An additional embodiment of the invention relates - to the administration of a pharmaceutical composition containing PAKl in conjunction with a pharmaceutically acceptable carrier, for any of the therapeutic effects discussed above.
  • Such pharmaceutical compositions may consist of PAKl, antibodies to PAKl, and mimetics, agonists, antagonists, or inhibitors of PAKl.
  • the compositions may be administered alone or in. combination with at least one other agent, such as a stabilizing compound, which may be administered in any sterile, biocompatible pharmaceutical canier including, but not limited to, saline, buffered saline, dextrose, and water.
  • the compositions may be administered to a patient alone, or in combination with other agents, drugs or hormones.
  • a pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration.
  • routes of administration include parenteral, e.g., infravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal, and rectal administration.
  • Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide.
  • the parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
  • compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions.
  • suitable carriers include physiological saline, bacteriostatic water, Cremophor EMTM (BASF, Parsippany, NJ.) or phosphate buffered saline (PBS).
  • the composition must be sterile and should be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi.
  • the canier can be a solvent or dispersion medium containing, for example, water, ethanol, a pharmaceutically acceptable polyol like glycerol, propylene glycol, liquid polyetheylene glycol, and suitable mixtures thereof.
  • the proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chloro- butanol, phenol, ascorbic acid, thimerosal, and the like.
  • isotonic agents for example, sugars, ⁇ olyalcohols such as mannitol, sorbitol, sodium chloride in the composition.
  • Prolonged abso ⁇ tion of the injectable compositions can be brought about by including in the composition an agent which delays abso ⁇ tion, for example, aluminum mo ' nostearate and gelatin.
  • Sterile injectable solutions can be prepared by inco ⁇ orating the active compound (e.g., a polypeptide or antibody) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization.
  • dispersions are prepared by inco ⁇ orating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above.
  • a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above.
  • the prefened methods of preparation are vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile- filtered solution thereof.
  • Oral compositions generally include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be inco ⁇ orated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and expectorated or swallowed.
  • compositions can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or com starch; a lubricant such as magne ' sium stearate or sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as 'sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.
  • a binder such as microcrystalline cellulose, gum tragacanth or gelatin
  • an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or com starch
  • a lubricant such as magne ' sium stearate or sterotes
  • a glidant such as colloidal
  • the compounds are delivered in the form of an aerosol spray from a pressurized container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.
  • a suitable propellant e.g., a gas such as carbon dioxide, or a nebulizer.
  • Systemic administration can also be by transmucosal or transdermal means.
  • penetrants appropriate to the barrier to be permeated are used in the formulation.
  • penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salt ' s, and fusidic acid derivatives.
  • Transmucosal administration can be accomplished through the use of nasal sprays or suppositories.
  • the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art.
  • the compounds can also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and 'other glycerides) or retention enemas for rectal delivery.
  • suppositories e.g., with conventional suppository bases such as cocoa butter and 'other glycerides
  • retention enemas for rectal delivery.
  • the active compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems.
  • a controlled release formulation including implants and microencapsulated delivery systems.
  • Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art.
  • the materials can also be obtained commercially from Alza Co ⁇ oration and Nova Pharmaceuticals, Inc.
  • Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to- viral antigens) can also be used as pharmaceutically acceptable caniers. These can be prepared according to methods known to those skilled in the art, for example, as described in US'. 4,522,811.
  • Dosage unit form refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
  • the specification for the dosage unit forms of the invention are dictated by and directly dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of individuals.
  • compositions can be included in a container, pack, or dispenser together with instructions for administration.
  • instructions for administration will specify use of the composition for cardiovascular diseases, dermatological diseaes, endocrinological diseases, metabolic diseases, inflammation, gastroenterological diseases, cancer, hematological diseases, respiratory diseases, muscle-skeleton diseases, neurological diseases and urological diseases.
  • compositions which include an agonist of PAKl activity, a compound which increases expression of PAKl, or a compound which increases expression or activity of a protein in the PAKl signaling pathway or any combination thereof
  • the instructions for administration will specify use of the composition for cardiovascular diseases, dermatological diseaes, endocrinological diseases, metabolic diseases, inflammation, gastroenterological diseases, cancer ' , hematological diseases, respiratory diseases, muscle-skeleton diseases, neurological diseases and urological diseases.
  • antibodies which specifically bind PAKl may be used, for the diagnosis of disorders characterized by the expression of PAKl, or in assays to monitor patients being treated with PAKl or agonists, antagonists, and inhibitors of PAKl.
  • Antibodies useful for diagnostic pu ⁇ oses may be prepared in the same manner as those described above for therapeutics. Diagnostic assays for PAKl include methods which utilize the antibody and a label to detect PAKl in human body fluids or in extracts of cells or tissues.
  • the antibodies may be used with or without modification, and may be labeled by covalent or non-covalent joining with a reporter molecule.
  • a wide variety of reporter molecules, several of which are described above, are known in the art and may be used.
  • a variety of protocols for measuring PAKl including ELISAs, RIAs, and FACS, are known in the art and provide a basis for diagnosing altered or abnormal levels of PAKl expression.
  • Normal or standard values for PAKl expression are established by combining body fluids or cell extracts taken from normal mammalian subjects, preferably human, with antibody to PAKl under conditions suitable for complex formation. The amount of standard complex formation may be quantified by various methods, preferably by photometric means. Quantities of PAKl expressed in subject samples from biopsied tissues are ' compared with the standard values. Deviation between standard and subject values establishes the parameters for diagnosing disease.
  • the polynucleotides encoding PAKT may be used for diagnostic pu ⁇ oses.
  • the polynucleotides which may be used include oligonucleotide sequences, complementary RNA and DNA molecules, and PNAs.
  • the polynucleotides may be used to detect and quantitate gene expression in biopsied tissues in which expression of PAKL 1 may be conelated with disease.
  • the diagnostic assay may be used to distinguish between absence, presence, and excess expression of PAKl, and to monitor regulation of PAKl levels during therapeutic intervention.
  • Polynucleotide sequences encoding PAKl may be used for the diagnosis of cardiovascular diseases, dermatological diseaes, endocrinological diseases, metabolic diseases, inflammation, gastroenterological diseases, cancer, hematological diseases, respiratory diseases, muscle-skeleton diseases, neurological diseases and urological diseases associated with expression of PAKl.
  • the polynucleotide sequences encoding PAKl may be used in Southern, Northern, or dot-blot analysis, or other membrane-based technologies; in PCR technologies; in dipstick, pin, and ELISA assays; and in microanays utilizing fluids or tissues from patient biopsies to detect altered PAKl expression. Such qualitative or quantitative methods are well known in the art.
  • nucleotide sequences encoding PAKl may be useful in assays that detect the presence of associated disorders, particularly those mentioned above.
  • the nucleotide sequences encoding PAKl may be labelled by standard methods and added to a fluid or tissue sample from a patient under conditions suitable for the formation of hybridization complexes. After a suitable incubation period, the sample is washed and the signal is quantitated and compared with a standard value.
  • nucleotide sequences have hybridized with nucleotide sequences in the sample, and the presence of altered levels of nucleotide sequences encoding PAKl in the sample indicates the presence of the associated disorder.
  • assays may also be used to evaluate the efficacy of a particular therapeutic treatment regimen in animal studies, in clinical trials, or in monitoring the treatment of an individual patient.
  • a normal or standard profile for expression is established. This may be accomplished by combining body fluids or cell extracts taken from normal subjects, either animal or human, with a sequence, or a fragment thereof, encoding PAKl, under conditions suitable for hybridization or amplification. Standard hybridization may be quantified by comparing the values obtained from normal subjects with values from an experiment in which a known amount of a substantially purified polynucleotide is used. Standard values obtained from normal samples may be compared with values obtained from samples from patients who are symptomatic for a disorder. Deviation from standard values is used to establish the presence of a disorder.
  • a therapeutically effective dose refers to that amount of active ingredient which increases or decreases PAKl activity relative to PAKl activity which occurs in the absence of the therapeutically effective dose.
  • the therapeutically effective dose can be estimated initially either in cell culture assays or in animal models, usually mice, rabbits, dogs, or pigs. The animal model also can be used to deterrnine the appropriate concentration range and route of administration. Such information can then be used to determine useful doses and routes for adminisfration in humans.
  • Therapeutic efficacy and toxicity can be determined by standard pharmaceutical procedures in cell cultures or experimental animals.
  • the dose ratio of toxic to therapeutic effects is the therapeutic index, and it can be expressed as the ratio, LD 50 /ED 50 -
  • Pharmaceutical compositions which exhibit large therapeutic indices are prefened.
  • the data obtained from cell culture assays and animal studies. is used in formulating a range of dosage for human use.
  • the dosage contained in such compositions is preferably within a range of circulating concentrations that include the ED 50 with little or no toxicity. The dosage varies within this range depending upon the dosage form employed, sensitivity of the patient, and the route of administration.
  • Dosage and administration are adjusted to provide sufficient levels of the active ingredient or to maintain the desired effect. Factors which can be taken into account include the severity of the disease state, general health of the subject, age, weight, and gender of the subject, diet, time and frequency of administration, d g combination(s), reaction sensitivities, and tolerance/response to therapy.
  • Long-acting pharmaceutical compositions can be administered every 3 to 4 days, every week, or once every two weeks depending on the half-life and clearance rate of the particular formulation.
  • Normal dosage amounts can vary from 0.1 micrograms to 100,000 micrograms, up to a total dose of about 1 g, depending upon the route of administration.
  • Guidance as to particular dosages and methods of delivery is provided in the literature and generally available to practitioners in the art. Those skilled in the art will employ different formulations for nucleotides than for proteins or their inhibitors. Similarly, delivery of polynucleotides or polypeptides will be specific to particular cells, conditions, locations, etc.
  • the reagent is a single-chain antibody
  • polynucleotides encoding the antibody can be constructed and introduced into a cell either ex vivo or in vivo using well- established techniques including, but not limited to, fransferrin-polycation-mediated DNA transfer- transfection with naked or encapsulated nucleic acids, liposome-mediated cellular fusion, intracellular transportation of DNA-coated latex beads, protoplast fusion, viral infection- electroporation, "gene gun", and DEAE- or calcium phosphate-mediated transfection.
  • the expression product is mRNA
  • the reagent is preferably an antisense oligonucleotide or a ribozyme.
  • Polynucleotides which express antisense oligonucleotides or ribozymes can be introduced into cells by a variety of methods, as described above.
  • a reagent reduces expression of PAKl gene or the activity of PAKl by at least about 10, preferably about 50, more preferably about 75, 90, or 100% relative to the absence of tfcie reagent.
  • the effectiveness of the mechanism chosen to decrease the level of expression of PAE 1 gene or the activity of PAKl can be assessed using methods well known in the art, such as hybridization of nucleotide probes to PAKl -specific mRNA, quantitative RT-PCR, immunologic detection of PAKl, or measurement of PAKl activity.
  • any of the pharmaceutical compositions of the invention can be administered in combination with other appropriate therapeutic agents. Selection of the appropriate agents for use in combination therapy can be made by one of ordinary skill in the art, according to conventional pharmaceutical principles.
  • the combination of therapeutic agents can act synergistically to effect the treatment or p-revention of the various disorders described above. Using this approach, one may be able to acbieve therapeutic efficacy with lower dosages of each agent, thus reducing the potential for adverse side effects.
  • Any of the therapeutic methods described above can be applied to any subject in need of such therapy, including, for example, mammals such as dogs, cats, cows, horses, rabbits, monkeys, and most preferably, humans.
  • Nucleic acid molecules of the invention are those nucleic acid molecules which are contained in a group of nucleic acid molecules consisting of (i) nucleic acid molecules encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 2, (ii) nucleic acid molecules comprising the sequence of SEQ ID NO: 1, (iii) nucleic acid molecules having the sequence of SEQ ID NO: 1, (iv)nucleic acid molecules the complementary strand of which hybridizes under stringent conditions to a nucleic acid molecule of (i), (ii), or (iii); and (v) nucleic acid molecules the sequence of which differs from the sequence of a nucleic acid molecule of (iii) due to the degeneracy of the genetic code, wherein the polypeptide encoded by said nucleic acid molecule has PAKl activity.
  • Polypeptides of the invention are those polypeptides, which are contained in a group of poly- peptides consisting of (i) polypeptides having the sequence of SEQ ID NO: 2, (ii) polypeptides comprising the sequence of SEQ ID NO: 2, (iii) polypeptides encoded by nucleic acid molecules of the invention and (iv) polypeptides which show at least 99%, 98%, 95%, 90%, or 80% homology with a polypeptide of (i), (ii), or (iii), wherein said purified polypeptide has PAKl activity.
  • An object of the invention is a method of screening for tberapeutic agents useful in the freatment of a disease comprised in a group of diseases consisting of " cardiovascular diseases, dermatological diseaes, endocrinological diseases, metabolic diseases, inflammation, gastroenterological diseases, cancer, hematological diseases, respiratory diseases, rnuscle-skeleton diseases, neurological diseases and urological diseases in a mammal comprising the steps of (i) contacting a test compound with a PAKl polypeptide, (ii) detect binding of said test compound to said PAKl polypeptide.
  • compounds that bind to the PAK 1 polypeptide are identified potential therapeutic agents for such a disease.
  • Another object of the invention is a method of screening for therapeutic agents useful in the treatment of a disease comprised in a group of diseases consisting of cardiovascular diseases, dermatological diseaes, endocrinological diseases, metabolic diseases, inflammation, gastroenterological diseases, cancer, hematological diseases, respiratory diseases, muscle-skeleton diseases, neurological diseases and urological diseases int a mammal comprising the steps of (i) determining the activity of a PAKl polypeptide at a certain concentration of a test compound or in the absence of said test compound, (ii) determining the activity of said polypeptide at a different concentration of said test compound.
  • compounds that lead to a difference in the activity of the PAKl polypeptide in (i) and (ii) are identified potential therapeutic agents for such a disease.
  • Another object of the invention is a method of screening for therapeutic agents useful in the freatment of a disease comprised in a group of diseases consisting of cardiovascular diseases, dermatological diseaes, endocrinological diseases, metabolic diseases, inflammation, gastroenterological diseases, cancer, hematological diseases, respiratory diseases, muscle-skeleton diseases, neurological diseases and urological diseases in- a mammal comprising the steps of (i) determining the activity of a PAKl polypeptide at a certain concentration of a test compound, (ii) determining the activity of a PAKl polypeptide at the pxesence of a compound known to be a regulator of a PAKl polypeptide.
  • compounds that sbow similar effects on the activity of the PAKl polypeptide in (i) as compared to compounds used i-n (ii) are identified potential therapeutic agents for such a disease.
  • test compound displaces a ligand which is first bound to the polypeptide.
  • Another object of the invention is a method of screening for therapeutic agents useful in the freatment of a disease comprised in a group of diseases consisting odf cardiovascular diseases, dermatological diseaes, endocrinological diseases, metabolic diseases, inflammation, gastroenterological diseases, cancer, hematological diseases, respiratory cdiseases, muscle-skeleton diseases, neurological diseases and urological diseases in a mammal comprising the steps of (i) contacting a test compound with a PAKl polynucleotide, (ii) detect bin -ing of said test compound to said PAKl polynucleotide.
  • Compounds that, e.g., bind to the PAKl polynucleotide are potential therapeutic agents for the treatment of such diseases.
  • Another object of the invention is the method of the above, wherein the nucleic acid molecule is RNA.
  • Another object of the invention is a method of the above, wherein the contacting step is in or at the surface of a cell.
  • Another object of the invention is a method of the above, wherein the contacting step is in a cell- free system.
  • Another object of the invention is a method of the above, wherein the polynucleotide is coupled to a detectable label.
  • Another object of the invention is a method of the above, wherein the test compound is coupled to a detectable label.
  • Another object of the invention is a method of diagnosing a disease comprised in a group of diseases consisting of cardiovascular diseases, dermatological diseaes, endocrinological diseases, metabolic diseases, inflammation, gastroenterological diseases, cancer, hematological diseases, respiratory diseases, muscle-skeleton diseases, neurological diseases and urologica-1 diseases in a mammal comprising the steps of (i) determining the amount of a PAKl polynucleotide in a sample taken from said mammal, (ii) determining the amount of PAKl polynucleotide in healthy and/or diseased mammal.
  • a disease is diagnosed, e.g., if there is a substantial similarity in- the amount of PAKl polynucleotide in said test mammal as compared to a diseased mammal.
  • Another object of the invention is a pharmaceutical composition for the treatment of a disease comprised in a group of diseases consisting of cardiovascular diseases, dermatological diseaes, endocrinological diseases, metabolic diseases, inflammation, gasfroenterological diseases, cancer, hematological diseases, respiratory diseases, muscle-skeleton diseases, neurological diseases and urological diseases in a mammal comprising a therapeutic agent which binds to a PAKl polypeptide.
  • Another object of the invention is a pharmaceutical composition for the freatment of a disease comprised in a group of diseases consisting of cardiovascular diseases, dermatological diseaes, endocrinological diseases, metabolic diseases, inflammation, gastroenterological diseases, cancer, hematological diseases, respiratory diseases, muscle-skeleton diseases, neurological diseases and urological diseases in a mammal comprising a therapeutic agent which regulates thie activity of a PAKl polypeptide.
  • Another object of the invention is a pharmaceutical composition for the treatment of a disease comprised in a group of diseases consisting of cardiovascular diseases, dermatological diseaes, endocrinological diseases, metabolic diseases, inflammation, gastroenterological diseases, cancer, hematological diseases, respiratory diseases, muscle-skeleton diseases, neurological diseases and urological diseases in a mammal comprising a therapeutic agent which regulates tl e activity of a PAKl polypeptide, wherein said therapeutic agent, is (i) a small molecule, (ii) an K NA molecule, (iii) an antisense oligonucleotide, (iv) a polypeptide, (v) an antibody, or (vi) a ribozytme.
  • cardiovascular diseases consisting of cardiovascular diseases, dermatological diseaes, endocrinological diseases, metabolic diseases, inflammation, gastroenterological diseases, cancer, hematological diseases, respiratory diseases, muscle-skeleton diseases, neurological diseases and urological diseases in a mammal
  • Another object of the invention is a pharmaceutical composition for the treatment of a disease comprised in a group of diseases consisting of cardiovascular diseases, dermatological diseaes, endocrinological diseases, metabolic diseases, inflammation, gastroenterological diseases, cancer, hematological diseases, respiratory diseases, muscle-skeleton diseases, neurological diseases and urological diseases in a mammal comprising a PAKl polynucleotide.
  • Another object of the invention is a pha ⁇ naceutical composition for the treatment of a disease comprised in a group of diseases consisting of cardiovascular diseases, dermatological diseaes, endocrinological diseases, metabolic diseases, inflammation, gasfroenterological diseases, cancer, hematological di._x.ases, respiratory diseases, muscle-skeleton diseases, neurological diseases and urological diseases in a mammal comprising a PA-vl polypeptide.
  • Another object of the invention is the use of regulators of a PAKl for the preparation of a pharmaceutical composition for the treatment of a disease comprised in a group of diseases consisting of cardiovascular diseases, dermatological diseaes, endocrinological diseases, metabolic diseases, inflammation, gastroenterological diseases, cancer, hematological diseases, respiratory diseases, muscle-skeleton diseases, neurological diseases and urological diseases in a mammal.
  • a group of diseases consisting of cardiovascular diseases, dermatological diseaes, endocrinological diseases, metabolic diseases, inflammation, gastroenterological diseases, cancer, hematological diseases, respiratory diseases, muscle-skeleton diseases, neurological diseases and urological diseases in a mammal.
  • Another object of the invention is a method for the preparation of a pharmaceutical composition useful for the treatment of a disease comprised in a group of diseases consisting of cardiovascular diseases, dermatological diseaes, endocrinological diseases, metabolic diseases, infla-tnmation, gasfroenterological diseases, cancer, hematological diseases, respiratory diseases, muscle-skeleton diseases, neurological diseases and urological diseases in a mammal comprising the steps of (i) identifying a regulator of PAKl, (ii) determining whether said regulator ameliorates the symptoms of a disease comprised in a group of diseases consisting of cardiovascular diseases, dermatological diseaes, endocrinological diseases, metabolic diseases, inflammation, gastroenterological diseases, cancer, hematological diseases, respiratory diseases, muscle-skeleton diseases, neixrological diseases and urological diseases in a mammal; and (iii) combining of said regulator with an acceptable pharmaceutical canier.
  • a group of diseases consisting of cardiovascular diseases, dermatological diseaes, end
  • Another object of the invention is the use of a regulator of PAKl for the regulation of PAKl activity in a mammal having a disease comprised in a group of diseases consisting of cardiovascular diseases, dermatological diseaes, endocrinological diseases, metabolic diseases, inflammation, gastroenterological diseases, cancer, hematological diseases, respiratory diseases, muscle-skeleton diseases, neurological diseases and urological diseases.
  • a regulator of PAKl for the regulation of PAKl activity in a mammal having a disease comprised in a group of diseases consisting of cardiovascular diseases, dermatological diseaes, endocrinological diseases, metabolic diseases, inflammation, gastroenterological diseases, cancer, hematological diseases, respiratory diseases, muscle-skeleton diseases, neurological diseases and urological diseases.
  • the degree of homology can readily be calculated by known methods. Prefened methods to determine homology are designed to give the largest match between the sequences tested. Methods to determine homology are codified in publicly available computer programs such as BestFit, BLASTP, BLASTN, and FASTA. The BLAST programs are publicly available from NCBI and other sources in the internet.
  • PAKl activated protein kinase
  • PAK 1 (p21 -activated kinase 1) (PAK-1) (P68-PAK) (Alpha-PAK) (Protein kinase MUK2) gb
  • PAK 1 p21 -activated kinase 1 (PAK-1) (P65-PAK) (Alpha-PAK) (CDC42/RAC effector kinase PAK-A) gb
  • RNA from each cell or tissue source was first reverse transcribed. 85 ⁇ g of total -RNA was reverse transcribed using 1 ⁇ mole random hexamer primers, 0.5 mM each of dATP, dCTP, dGTP and dTTP (Qiagen, Hilden, Germany), 3000 U RnaseQut (Invitrogen, Groningen, Netherlands) in a final volume of 680 ⁇ l.
  • the first strand synthesis buffer and Omniscript reverse transcriptase (2 u/ ⁇ l) were from (Qiagen, Hilden, Germany). The reaction was incubated at 37°C for 90 minutes and cooled on ice. The volume was adjusted to 6800 ⁇ l with water, yielding a final concentration of 12.5 ng/ ⁇ l of starting RNA.
  • the PAKl forward primer sequence was: Primerl (SEQ ID NO: 3).
  • the PAKl reverse primer sequence was Primer2 (SEQ ID NO: 4).
  • Probel SEQ ID NO: 5
  • FAM carboxyfluorescein succinimidyl ester
  • TAMRA carboxytetramethylrhodamine
  • the following reagents were prepared in a total of 25 ⁇ l : lx TaqMan buffer A, 5.5 mM MgCl 2 , 200 nM of dATP, dCTP, dGTP, and dUTP, 0.025 U/ ⁇ AmpliTaq GoldTM, 0.01 U/ ⁇ l AmpErase and Probel (SEQ ID NO: 5), PAKl forward and reverse primers each at 200 nM, 200 nM PAKl FAM/TAMRA-labelled probe, and 5 ⁇ l of template cDNA.
  • Thermal cycling parameters were 2 min at 50°C, followed by 10 min at 95°C, followed by 40 cycles of melting at 95°C for 15 sec and annealing/extending at 60°C for 1 min.
  • the CT (threshold cycle) value is calculated as described in the "Quantitative determination of nucleic acids" section.
  • the CF-value (factor for • threshold cycle conection) is calculated as follows :
  • PCR reactions were set up to quantitate the housekeeping genes (HKG) for each cDNA sample.
  • CT ⁇ G -values were calculated as described in the "Quantitative determination of nucleic acids" section.
  • CT cDNA - n CT value of the tested gene .for the cDNA n
  • CF cDNA - n detection factor for cDNA n
  • PAKl The expression of PAKl was investigated in the tissues in table 1.
  • esophagus 111 esophagus tumor 290 stomach 1746 stomach tumor 98 colon 419 colon tumor 40 small intestine 3148 ileum 288 ileum tumor 22 ileum chronic inflammation 85 rectum 1105 salivary gland 3350 fetal liver 91 liver 83 liver liver cinhosis 1389 liver lupus disease 17 liver tumor 116 HEP G2 cells 2
  • HeLa cells 55 placenta 100 uterus 45 uterus tumor 31 ovary 2435 ovary tumor 118 breast 35 breast tumor 350
  • MDA MB 231 cells (breast tumor) 28 mammary gland 111
  • cDNA sequence coding for PAKl enables its use as a tool for antisense technology in the investigation of gene function.
  • Oligonucleotides, cDNA or genomic fragments comprising the antisense strand of a polynucleotide coding for PAKl are used either in vitro or in vivo to inhibit translation of the mRNA.
  • antisense molecules can be designed at various locations along the nucleotide sequences.
  • the gene of interest is effectively turned off.
  • the function of the gene is ascertained by observing behavior at the intracellular, cellular, tissue or organismal level (e.g., lethality, loss of differentiated function, changes in mo ⁇ hology, etc.).
  • modifications of gene expression is obtained by designing antisense sequences to intron regions, promoter/enhancer elements, or even to trans-acting regulatory genes.
  • Expression of PAKl is accomplished by subcloning the cDNAs into appropriate expression vectors and transfecting the vectors into expression hosts such as, e.g., E. coli. m a particular case, the vector is engineered such that it contains a promoter for ⁇ -galactosidase, upstream of the cloning site, followed by sequence containing the amino-terminal Methionine and the subsequent seven residues of ⁇ -galactosidase. Immediately following these eight residues is an engineered bacteriophage promoter useful for artificial priming and transcription and for providing a number of unique endonuclease restriction sites for cloning.
  • IPTG Isopropyl- ⁇ -D-thiogalactopyranoside
  • the cDNA is not in the proper reading frame, it is obtained by deletion or insertion of the appropriate number of bases using well known methods including in vitro, mutagenesis, digestion with exonuclease DI or mung bean nuclease, or the inclusion of an oligonucleotide linker of appropriate length.
  • the PAKl cDNA is shuttled into other vectors known to be useful for expression of proteins in specific hosts.
  • Oligonucleotide primers containing cloning sites as well as a segment of DNA (about 25 bases) sufficient to hybridize to stretches at both ends of the target cDNA is synthesized chemically by standard methods. These primers' are then used to amplify the desired gene segment by PCR. The resulting gene segment is digested with appropriate restriction enzymes under standard conditions and isolated by gel electrophoresis. Alternately, similar gene segments are produced by digestion of the cDNA with appropriate restriction enzymes. Using appropriate primers, segments of coding sequence from more than one gene are ligated together and cloned in appropriate vectors. It is possible to optimize expression by construction of such chimeric sequences.
  • Suitable expression hosts for such chimeric molecules include, but are not limited to, mammalian cells such as Chinese Hamster Ovary (CHO) and human 293 cells., insect cells such as S£9 cells, yeast cells such as Saccharomyces cerevisiae and bacterial cells such as E. coli.
  • a useful expression vector also includes an origin of replication to allow propagation in bacteria, and a selectable marker such as the ⁇ -lactamase antibiotic resistance gene to allow plasmid selection in bacteria.
  • the vector may include a second selectable marker such as the neomycin phosphotransferase gene to allow selection in transfected eukaryotic host cells.
  • Vectors for use in eukaryotic expression hosts require RNA processing elements such as 3' polyadenylation sequences if such are not part of the cDNA of interest.
  • the vector contains promoters or enhancers which increase gene expression.
  • promoters are host specific and include MMTV, SV4 ⁇ , and meiallothionine promoters for CHO cells; frp, lac, tac and T7 promoters for bacterial hosts; and alpha factor, alcohol oxidase and PGH promoters for yeast.
  • Transcription enhancers such as ' the rous sarcoma vims enhancer, are used in mammalian host cells. Once homogeneous cultures of recombinant cells are obtained through standard culture methods, large quantities of recombinantly produced PAKl are recovered from the conditioned medium and analyzed using chromatographic methods known in the art.
  • PAKl can be cloned into the expression vector pcDNA3, as exemplified herein.
  • This product can be used to transform, for example, HEK293 or COS by methodology standard in the art. Specifically, for example, using Lipofeetamine (Gibco BRL catalog no. 18324-020) mediated gene transfer.
  • Example 5 Isolation of Recombinant P A K1
  • PAKl is expressed as a chimeric protein with one. or more additional polypeptide domains added to facilitate protein purification.
  • purification facilitating domains include, but are not limited to, metal chelating peptides such as histidine-tryptophan modules that allow purification on immobilized metals [Appa Rao, 1997] and the domain utilized in the FLAGS extension/affinity purification system (Immunex Co ⁇ ., Seattle, Washington).
  • the inclusion of a cleavable linker sequence such as Factor Xa or enterokinase (Invitrogen, Groningen, The Netherlands) between the purification domain and the PAKl sequence is useful to facilitate expression of PAKl.
  • the following example provides a method for purifying PAKl .
  • PAKl is generated using the baculovirus expression system BAC-TO-BAC (GIBCO BRL) based on Autographa californica nuclear polyhedrosis virus (AcNPV) infection of Spodoptera frugiperda insect cells (Sf cells).
  • the recombinant plasmid is transformed into DH10BAC competent cells which contain the parent b'acmid bMON14272 (AcNPV infectious DNA) and a helper plasmid.
  • the mini-Tn7 element on the pFASTBAC donor can transpose to the attTn7 attachment site on the bacmid thus introducing the kinase gene into the viral genome. Colonies containing recombinant bacmids are identified by disruption of the lacZ gene.
  • the kinase/bacmid construct can then be isolated and infected into insect cells (Sf9 cells) resulting in the production of infectious recombinant baculovirus particles and expression of either unfused recombinant enzyme (pFastbacl) or PAKl-His fusion protein (pFastbacHT).
  • pFastbacl unfused recombinant enzyme
  • pFastbacHT PAKl-His fusion protein
  • denatured protein from reverse phase HPLC separation is obtained in quantities up to 75 mg. This denatured protein is used to immunize mice or rabbits using standard protocols; about 100 ⁇ g are adequate for immunization of a mouse, while up to 1 mg might be used to immunize a rabbit.
  • the denatured protein is radioiodinated and used to screen potential murine B- cell hybridomas for those which produce antibody;- This procedure requires only small quantities of protein, such that 20 mg is sufficient for labeling and screening of several thousand clones.
  • the amino acid sequence of an appropriate PAKl domain is analyzed to determine regions of high antigenicity.
  • Oligopeptides comprising appropriate hydrophihc regions are synthesized and used in suitable immunization protocols to raise antibodies.
  • the optimal amino acid sequences for immunization are usually at the C-terminus, the N-terminus and those intervening, hydrophihc regions of the polypeptide which are likely to be exposed to the external environment when the protein is in its natural conformation.
  • selected peptides typically, about 15 residues in length, are synthesized using an Applied Biosystems Peptide Synthesizer Model 431 A using fmoc-chemistry and coupled to keyhole limpet hemocyanin (KLH; Sigma, St. Louis, MO) ' by reaction with M-maleimidobenzoyl-N-hydroxy- succinimide ester, MBS. If necessary, a cysteine is introduced at the N-terminus of the peptide to permit coupling to KLH. Rabbits are immunized with the peptide-KLH complex in complete Freund's adjuvant.
  • KLH keyhole limpet hemocyanin
  • the resulting antisera are tested for antipeptide activity by binding the peptide to plastic, blocking with 1% bovine serum albumin, reacting with antisera, washing and reacting with labeled (radioactive or fluorescent), affinity purified, specific goat anti-rabbit IgG.
  • Hybridomas are prepared and screened using standard techniques. Hybridomas of interest are detected by screening with labeled PAKl to identify those fusions producing the monoclonal antibody with the desired specificity.
  • wells of plates FAST; Becton- Dickinson, Palo Alto, CA
  • affinity purified, specific rabbit anti- mouse (or suitable antispecies 1 g) antibodies at 10 mg/ml.
  • the coated wells are blocked with 1% bovine serum albumin, (BSA), washed and incubated with supematants from hybridomas. After washing the wells are incubated with labeled PAKl at 1 mg/ml.
  • BSA bovine serum albumin
  • Supematants with specific antibodies bind more labeled PAKl than is detectable in the background. Then clones producing specific antibodies are expanded and subjected to two cycles of cloning at limiting dilution. Cloned hybridomas are injected into pristane-treated mice to produce ascites, and monoclonal antibody is purified from mouse ascitic fluid by affinity chromatography on Protein A. Monoclonal antibodies with affinities of at least
  • PAKl antibodies are useful for investigating signal transduction and the diagnosis of infectious or hereditary conditions which are characterized by differences in the amount or distribution of PAKl or downstream products of an active signaling cascade.
  • Diagnostic tests for PAKl include methods utilizing 'antibody and a label to detect PAKl in human body fluids, membranes, cells, tissues or extracts ⁇ f-such.
  • the polypeptides and antibodies of the present invention are used with or without modification. Frequently, the polypeptides and antibodies are labeled by joining them, either covalently or noncovalently, with a substance which provides for a detectable signal.
  • labels and conjugation techniques are known and have been reported extensively in both the scientific and patent literature. Suitable labels include radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent agents, chemi- luminescent agents, chromogenic agents, magnetic particles and the like.
  • a variety of protocols for measuring soluble or membrane-bound PA l, using either polyclonal or monoclonal antibodies specific for the protein, are known in the art. Examples include enzyme- linked immunosorbent assay (ELISA), radioimmunoassay (RIA) and fluorescent activated cell sorting (FACS).
  • ELISA enzyme- linked immunosorbent assay
  • RIA radioimmunoassay
  • FACS fluorescent activated cell sorting
  • a two-site monoclonal-based immunoassay utilizing monoclonal antibodies reactive to two non-interfering epitopes on PAKl is prefened, but a competitive binding assay may be employed.
  • Native or recombinant PAKl is purified by immunoaffinity chromatography using antibodies specific for PAKl.
  • an immunoaffinity column is constructed by covalently coupling the anti-TRH antibody to an activated chromatographic resin.
  • Polyclonal immunoglobulins are prepared from immune sera either by precipitation with ammonium sulfate or by purification on immobilized Protein A (Pharmacia LKB Biotechnology, Piscataway N.J.). Likewise, monoclonal antibodies are prepared from mouse ascites fluid by ammonium sulfate precipitation or chromatography on immobilized Protein A. Partially purified immunoglobulin is covalently attached to a chromatographic resin such as CnBr-activated Sepharose (Pharmacia LKB Biotechnology). The antibody is coupled to the resin, the resin is blocked, and the derivative resin is washed according to the manufacturer's instructions.
  • a chromatographic resin such as CnBr-activated Sepharose
  • Such immunoaffinity columns are utilized in the purification of PA l by preparing a fraction from cells containing PAKl in a soluble form. This preparation is derived by solubilization of whole cells or of a subcellular fraction obtained via differential centrifugation, (with or without addition of detergent) or by other methods well known in the art. Alternatively, soluble PAKl containing a signal sequence is secreted in useful quantity into the medium in which the cells are grown.
  • a soluble PAKl -containing preparation is passed over the immunoaffinity column, and the column is washed under conditions that allow the preferential absorbance of PAKl (e.g., high ionic strength buffers in the presence of detergent). Then, the column is eluted under conditions that disrupt antibody/protein binding (e.g., a buffer of pH 2-3 or a high concentration of a chaotrope such as urea or thiocyanate ion), and PAKl is collected.
  • a buffer of pH 2-3 or a high concentration of a chaotrope such as urea or thiocyanate ion
  • This invention is particularly useful for screening therapeutic compounds by using PAKl or fragments thereof in any of a variety of dmg screening techniques.
  • the following example provides a system for dmg screening measuring the kinase activity.
  • the recombinant kinase-His fusion protein can be purified from the crude lysate by metal-affinity chromatography using Ni-NTA agarose. This allows the specific retention of the recombinant material (since this is fused to the His-tag) whilst the endogenous insect proteins are washed off. The recombinant material is then eluted by competition with imidazol.
  • PAKl molecules of the present invention can be measured using a variety of assays that measure PAKl activity.
  • PAKl enzyme activity can be assessed by a standard in vitro kinase assay.
  • the kinase activity of the kinase can be detected, for example, by adding ATP having radioactively labeled phosphate to the reaction system containing the protein of the present invention and the substrate and measuring the radioactivity of the phosphate attached to the subsfrate.
  • dmg design is to produce structural analogs of biologically active polypeptides of interest or of small molecules with which they interact, agonists, antagonists, or inhibitors. Any of these examples are used to fashion d gs which are more active or stable forms of the polypeptide or which enhance or interfere with the function of a polypeptide in vivo.
  • the three-dimensional structure of a protein of interest, or of a protein-inhibitor complex is determined by x-ray crystallography, by computer modeling or, most typically, by a combination of the two approaches. Both the shape ' and charges of the polypeptide must be ascertained to elucidate the structure and to determine active site(s) of the molecule. Less often, useful information regarding the structure of a polypeptide is gained by modeling based on the structure of homologous proteins. In botb cases, relevant structural information is used to design efficient inhibitors. Useful examples of rational drug design include molecules which have improved activity or stability or which act as inhibitors, agonists, or antagonists of native peptides.
  • a target-specific antibody selected by functional assay, as described above, and then to solve its crystal structure.
  • This approach in principle, yields a pharmacore upon which subsequent dmg design is based. It is possible to bypass protein crystallography altogether by generating anti-idiotypic antibodies (anti-ids) to a functional, pharmacologically active antibody. As a minor image of a minor image, the binding site of the anti-ids is expected to be an analog of the original receptor. The ant ⁇ id is then used to identify and isolate peptides from banks of chemically or biologically produced peptides. The isolated peptides then act as the pharmacore.
  • anti-ids anti-idiotypic antibodies
  • Labeled PAKl is useful as a reagent for the purification of molecules with which it interacts.
  • P-AK1 is covalently coupled to a chromatography column.
  • Cell-free exfract derived from synovial cells or putative target cells is passed over the column, and molecules with appropriate affinity bind to PAKl.
  • PAKl -complex is recovered from the column, and the PAKl -binding ligand disassociated and subjected to N-terminal protein sequencing. The amino acid sequence information is then used to identify the captured molecule or to design degenerate oligonucleotide probes for cloning the relevant gene from an appropriate cDNA. library.
  • antibodies are raised against PAKl, specifically monoclonal antibodies.
  • the monoclonal antibodies are screened to identify those which inhibit the binding of " labeled PAKl. These monoclonal antibodies are then used therapeutically.
  • Example 12 Use and Administration of Antibodies, Inhibitors, or Antagonists
  • LSTs are formulated in a nontoxic, inert, pharmaceutically acceptable aqueous carrier medium preferably at a pH of about 5 to 8, more preferably 6 to 8, although pH may vary according to the characteristics of the antibody, inhibitor, or antagonist being formulated and the condition to be treated. Characteristics of LSTs include solubility of the molecule, its half-life and antigenicity/- immunogenicity. These and other characteristics aid in defining an effective carrier. Native human proteins are prefened as LSTs, but organic or synthetic molecules resulting from drug screens are equally effective in particular situations.
  • LSTs are delivered by known routes of adminisfration including but not limited to topical creams and gels; transmucosal spray and aerosol; transdermal patch and bandage; injectable, infravenous and lavage formulations; and orally administered liquids and pills particularly formulated to resist stomach acid and enzymes.
  • routes of adminisfration including but not limited to topical creams and gels; transmucosal spray and aerosol; transdermal patch and bandage; injectable, infravenous and lavage formulations; and orally administered liquids and pills particularly formulated to resist stomach acid and enzymes.
  • the particular formulation, exact dosage, and route of administration is determined by the attending physician and varies according to each specific situation.
  • Such determinations are made by considering multiple variables such as the condition to be treated, the LST to be administered, and the pharmacokinetic profile of a particular LST. Additional factors which are taken into account include severity of the disease state, patient's age, weight, gender and diet, time and frequency of LST administration, possible combination with other dmgs, reaction sensitivities, and tolerance/response to therapy. Long acting LST formulations might be administered every 3 to 4 days, every week, or once every two weeks depending on half-life and clearance rate of the particular LST.
  • Normal dosage amounts vary from 0.1 to IO 5 ⁇ g, up to a total dose of about 1 g, depending upon the route of adminisfration.
  • Guidance as to particular dosages and methods of delivery is provided in the literature; see U.S. Pat. Nos. 4,657,760; 5,206,344; or 5,225,212.
  • Those skilled in the art employ different formulations for different LSTs.
  • Administration to cells such as nerve cells necessitates delivery in a manner different from that to other cells such as vascular endothelial cells.
  • abnormal signal transduction, trauma, or diseases which trigger PAKl activity are treatable with LSTs. These conditions or diseases are specifically diagnosed by the tests discussed above, and such testing should be performed in suspected cases of viral, bacterial or fungal infections, allergic responses, mechanical injury associated with trauma, hereditary diseases, lymphoma or carcinoma, or other conditions which activate the genes of lymphoid or neuronal tissues.
  • Animal model systems which elucidate the physiological and behavioral roles of the PAKl are produced by creating nonhuman transgenic animals in which the activity of the PAKl is either increased or decreased, or the amino acid sequence of the e-xpressed PAKl is altered, by a variety of techniques.
  • Examples of these techniques include, but are not limited to: 1) Insertion of normal or mutant versions of DNA encoding a PAKl, by mic-roinjection, elecfroporation, retroviral transfection or other means well known to those skilled in the art, into appropriately fertilized embryos in order to produce a transgenic animal or 2) homologous recombination of mutant or normal, human or animal versions of these genes with the native gene locus in transgenic animals to alter the regulation of expression or the structure of these PAKl sequences.
  • the technique of homologous recombination is well known in the art.
  • One means available for producing a transgenic animal is as follows: Female mice are mated, and the resulting fertilized eggs are dissected out of their oviducts. The eggs are stored in an appropriate medium such as cesiumchloride M2 medium. DNA or cDNA encoding PAKl is purified from a vector by methods well known to the one skilled in the art.
  • Inducible promoters may be fused with the coding region of the DNA to provide an experimental means to regulate expression of the transgene.
  • tissue specific regulatory elements may be fused with the coding region to permit tissue-specific expression of the transgene.
  • microinjection is not the only method for inserting DNA into the egg but is used here only for exemplary pu ⁇ oses.

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Abstract

La présente invention a trait à une PAK1 humaine associée aux maladies cardio-vasculaires, aux maladies dermatologiques, aux maladies endocriniennes, aux maladies métaboliques, à l'inflammation (par exemple, la cirrhose du foie), aux maladies gastro-entérologiques, au cancer (par exemple, le cancer de l'estomac, du côlon, de l'ovaire ou de la prostate), aux maladies hématologiques, aux maladies respiratoires, aux maladies musculo-squelettiques, aux maladies neurologiques (par exemple, la maladie d'Alzheimer) et aux maladies urologiques. L'invention a également trait à des dosages pour l'identification de composés utiles dans le traitement des maladies cardio-vasculaires, des maladies dermatologiques, des maladies endocriniennes, des maladies métaboliques, de l'inflammation, des maladies gastro-entérologiques, du cancer, des maladies hématologiques, des maladies respiratoires, des maladies musculo-squelettiques, des maladies neurologiques et des maladies urologiques. L'invention a trait en outre à des composés de liaison et/ou d'activation ou d'inhibition de l'activité de PAK1 ainsi qu'à des compositions pharmaceutiques comportant de tels composés.
PCT/EP2005/001749 2004-03-04 2005-02-19 Agents de diagnostic et therapeutiques pour des maladies associees a proteine kinase p21/cdc42/rac1 WO2005095977A1 (fr)

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CN103913570A (zh) * 2014-03-24 2014-07-09 张灏 癌症危险分层生物标志物、其应用以及癌症危险分层装置
WO2020212484A1 (fr) * 2019-04-17 2020-10-22 INSERM (Institut National de la Santé et de la Recherche Médicale) Procédés et compositions de traitement de troubles dépendants de il-1beta mediés par inflamasome nlrp3
CN113398244A (zh) * 2021-05-28 2021-09-17 南方医科大学 一种治疗帕金森病的制剂及其应用

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US5952217A (en) * 1997-09-23 1999-09-14 Bristol-Myers Squibb Company Recombinant yeast cell and assay using same
WO2003103601A2 (fr) * 2002-06-05 2003-12-18 Metabolex, Inc. Methodes de diagnostic et de traitement du diabete et de la resistance a l'insuline
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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103913570A (zh) * 2014-03-24 2014-07-09 张灏 癌症危险分层生物标志物、其应用以及癌症危险分层装置
CN103913570B (zh) * 2014-03-24 2016-03-23 张灏 癌症危险分层生物标志物、其应用以及癌症危险分层装置
WO2020212484A1 (fr) * 2019-04-17 2020-10-22 INSERM (Institut National de la Santé et de la Recherche Médicale) Procédés et compositions de traitement de troubles dépendants de il-1beta mediés par inflamasome nlrp3
CN113398244A (zh) * 2021-05-28 2021-09-17 南方医科大学 一种治疗帕金森病的制剂及其应用
CN113398244B (zh) * 2021-05-28 2023-07-07 南方医科大学 一种治疗帕金森病的制剂及其应用

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