US20030013144A1 - HS2STs as modifiers of the p53 pathway and methods of use - Google Patents

HS2STs as modifiers of the p53 pathway and methods of use Download PDF

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US20030013144A1
US20030013144A1 US10/161,398 US16139802A US2003013144A1 US 20030013144 A1 US20030013144 A1 US 20030013144A1 US 16139802 A US16139802 A US 16139802A US 2003013144 A1 US2003013144 A1 US 2003013144A1
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hs2st
assay
agent
leu
candidate
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Lori Friedman
Gregory Plowman
Marcia Belvin
Helen Francis-Lang
Danxi Li
Roel Funke
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Exelixis Inc
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Assigned to EXELIXIS, INC. reassignment EXELIXIS, INC. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: FUNKE, ROEL P., BELVIN, MARCIA, FRANCIS-LANG, HELEN, LI, DANXI, FRIEDMAN, LORI, PLOWMAN, GREGORY D.
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Definitions

  • the p53 gene is mutated in over 50 different types of human cancers, including familial and spontaneous cancers, and is believed to be the most commonly mutated gene in human cancer (Zambetti and Levine, FASEB (1993) 7:855-865; Hollstein, et al., Nucleic Acids Res. (1994) 22:3551-3555). Greater than 90% of mutations in the p53 gene are missense mutations that alter a single amino acid that inactivates p53 function.
  • the human p53 protein normally functions as a central integrator of signals including DNA damage, hypoxia, nucleotide deprivation, and oncogene activation (Prives, Cell (1998) 95:5-8). In response to these signals, p53 protein levels are greatly increased with the result that the accumulated p53 activates cell cycle arrest or apoptosis depending on the nature and strength of these signals. Indeed, multiple lines of experimental evidence have pointed to a key role for p53 as a tumor suppressor (Levine, Cell (1997) 88:323-331). For example, homozygous p53 “knockout” mice are developmentally normal but exhibit nearly 100% incidence of neoplasia in the first year of life (Donehower et al., Nature (1992) 356:215-221).
  • Heparan sulphate (HS) proteoglycans are ubiquitous on cell surfaces and in the extracellular matrix, are composed of extended polysaccharide (glycosaminoglycan) chains covalently bound to various core proteins.
  • HS has been associated in a variety of biological processes, such as assembly of extracellular matrices, control of cellular growth and differentiation, regulation of blood coagulation, viral infection, etc. (Lindahl, U. et al. (1994) Thromb. Res. 75, 1-32) (Lindahl, U. et al. (1998) J. Biol. Chem. 273, 24979-24982) (Salmivirta, M., et al.
  • a precursor polysaccharide composed of alternating D-glucuronic acid (GlcA) and N-acetyl-D-glucosamine (GlcNAc; 2-deoxy-2-acetoamido-D-glucose) units is therefore modified through a series of reactions that include, in consecutive order, N-deacetylation deacetylation and N-sulphation of GlcN residues, C-5 epimerization of GlcA to L-iduronic iduronic acid (IdoA), 2-O-sulphation of uronic acid residues, and finally 6-0- and 3-O-sulphation sulphation of GlcN residues (Lindahl, U. et al. (1998) supra) (Salmivirta, M., et al. (1996)supra) (Rosenberg, R, et al. (1997) supra).
  • Heparan sulfate 2-O-sulfotransferase has been noted as a putative sulfotransferase enzyme that may play a role in heparan sulfate proteoglycan biosynthesis.
  • Uronyl 2-sulfotransferase (UST or DS2ST) is a closely related enzyme that contains sulfates iduronyl and glucuronyl that residues in dermatan/chondroitin sulfate (Kobayashi, M. et al (1997) J. Biol. Chem. 272, 13980-13985).
  • UST has ubiquitous expression of messages in a number of human tissues and in several human cancer cell lines (Kobayashi, M. et al. (1999) J Biol Chem 274, 10474-80).
  • a genetic screen can be carried out in an invertebrate model organism having underexpression (e.g. knockout) or overexpression of a gene (referred to as a “genetic entry point”) that yields a visible phenotype. Additional genes are mutated in a random or targeted manner.
  • the gene When a gene mutation changes the original phenotype caused by the mutation in the genetic entry point, the gene is identified as a “modifier” involved in the same or overlapping pathway as the genetic entry point.
  • the genetic entry point is an ortholog of a human gene implicated in a disease pathway, such as p53, modifier genes can be identified that may be attractive candidate targets for novel therapeutics.
  • HS2ST genes that modify the p53 pathway in Drosophila, and identified their human orthologs, hereinafter referred to as HS2ST.
  • the invention provides methods for utilizing these p53 modifier genes and polypeptides to identify candidate therapeutic agents that can be used in the treatment of disorders associated with defective p53 function.
  • Preferred HS2ST-modulating agents specifically bind to HS2ST polypeptides and restore p53 function.
  • Other preferred HS2ST-modulating agents are nucleic acid modulators such as antisense oligomers and RNAi that repress HS2ST gene expression or product activity by, for example, binding to and inhibiting the respective nucleic acid (i.e. DNA or mRNA).
  • HS2ST-specific modulating agents may be evaluated by any convenient in vitro or in vivo assay for molecular interaction with an HS2ST polypeptide or nucleic acid.
  • candidate p53 modulating agents are tested with an assay system comprising a HS2ST polypeptide or nucleic acid.
  • Candidate agents that produce a change in the activity of the assay system relative to controls are identified as candidate p53 modulating agents.
  • the assay system may be cell-based or cell-free.
  • HS2ST-modulating agents include HS2ST related proteins (e.g.
  • a small molecule modulator is identified using a transferase assay.
  • the screening assay system is selected from a binding assay, an apoptosis assay, a cell proliferation assay, an angiogenesis assay, and a hypoxic induction assay.
  • candidate p53 pathway modulating agents are further tested using a second assay system that detects changes in the p53 pathway, such as angiogenic, apoptotic, or cell proliferation changes produced by the originally identified candidate agent or an agent derived from the original agent.
  • the second assay system may use cultured cells or non-human animals.
  • the secondary assay system uses non-human animals, including animals predetermined to have a disease or disorder implicating the p53 pathway, such as an angiogenic, apoptotic, or cell proliferation disorder (e.g. cancer).
  • the invention further provides methods for modulating the p53 pathway in a mammalian cell by contacting the mammalian cell with an agent that specifically binds a HS2ST polypeptide or nucleic acid.
  • the agent may be a small molecule modulator, a nucleic acid modulator, or an antibody and may be administered to a mammalian animal predetermined to have a pathology associated the p53 pathway.
  • HS2ST-modulating agents that act by inhibiting or enhancing HS2ST expression, directly or indirectly, for example, by affecting an HS2ST function such as enzymatic (e.g., catalytic) or binding activity, can be identified using methods provided herein.
  • HS2ST modulating agents are useful in diagnosis, therapy and pharmaceutical development.
  • Genbank referenced by Genbank identifier (GI) number
  • Genbank identifier GI#s 6683563 (SEQ ID NO:1), 12545388 (SEQ ID NO:2), and 4803734 (SEQ ID NO:5) for nucleic acid
  • GI#s 6683564 SEQ ID NO:6
  • 6912420 SEQ ID NO:7
  • 4803735 SEQ ID NO:8
  • 5032219 SEQ ID NO:9
  • HS2STs are sulfotransferase proteins with transferase domains.
  • the term “HS2ST polypeptide” refers to a full-length HS2ST protein or a functionally active fragment or derivative thereof.
  • a “functionally active” HS2ST fragment or derivative exhibits one or more functional activities associated with a full-length, wild-type HS2ST protein, such as antigenic or immunogenic activity, enzymatic activity, ability to bind natural cellular substrates, etc.
  • HS2ST proteins, derivatives and fragments can be assayed by various methods known to one skilled in the art (Current Protocols in Protein Science (1998) Coligan et al., eds., John Wiley & Sons, Inc., Somerset, N.J.) and as further discussed below.
  • functionally active fragments also include those fragments that comprise one or more structural domains of an HS2ST, such as a binding domain. Protein domains can be identified using the PFAM program (Bateman A., et al., Nucleic Acids Res, 1999, 27:260-2; http://pfam.wustl.edu). Methods for obtaining HS2ST polypeptides are also further described below.
  • preferred fragments are functionally active, domain-containing fragments comprising at least 25 contiguous amino acids, preferably at least 50, more preferably 75, and most preferably at least 100 contiguous amino acids of any one of SEQ ID NOs:6, 7, 8, or 9 (an HS2ST).
  • the fragment comprises the entire transferase (functionally active) domain.
  • HS2ST nucleic acid refers to a DNA or RNA molecule that encodes a HS2ST polypeptide.
  • the HS2ST polypeptide or nucleic acid or fragment thereof is from a human, but can also be an ortholog, or derivative thereof with at least 70% sequence identity, preferably at least 80%, more preferably 85%, still more preferably 90%, and most preferably at least 95% sequence identity with HS2ST.
  • orthologs in different species retain the same function, due to presence of one or more protein motifs and/or 3-dimensional structures. Orthologs are generally identified by sequence homology analysis, such as BLAST analysis, usually using protein bait sequences.
  • Sequences are assigned as a potential ortholog if the best hit sequence from the forward BLAST result retrieves the original query sequence in the reverse BLAST (Huynen M A and Bork P, Proc Natl Acad Sci (1998) 95:5849-5856; Huynen M A et al., Genome Research (2000) 10:1204-1210).
  • Programs for multiple sequence alignment such as CLUSTAL (Thompson J D et al, 1994, Nucleic Acids Res 22:4673-4680) may be used to highlight conserved regions and/or residues of orthologous proteins and to generate phylogenetic trees.
  • orthologous sequences from two species generally appear closest on the tree with respect to all other sequences from these two species.
  • Structural threading or other analysis of protein folding e.g., using software by ProCeryon, Biosciences, Salzburg, Austria
  • a gene duplication event follows speciation, a single gene in one species, such as Drosophila, may correspond to multiple genes (paralogs) in another, such as human.
  • the term “orthologs” encompasses paralogs.
  • percent (%) sequence identity with respect to a subject sequence, or a specified portion of a subject sequence, is defined as the percentage of nucleotides or amino acids in the candidate derivative sequence identical with the nucleotides or amino acids in the subject sequence (or specified portion thereof), after aligning the sequences and introducing gaps, if necessary to achieve the maximum percent sequence identity, as generated by the program WU-BLAST-2.0a19 (Altschul et al., J. Mol. Biol. (1997) 215:403-410; http://blast.wustl.edu/blast/README.html) with all the search parameters set to default values.
  • the HSP S and HSP S2 parameters are dynamic values and are established by the program itself depending upon the composition of the particular sequence and composition of the particular database against which the sequence of interest is being searched.
  • a % identity value is determined by the number of matching identical nucleotides or amino acids divided by the sequence length for which the percent identity is being reported. “Percent (%) amino acid sequence similarity” is determined by doing the same calculation as for determining % amino acid sequence identity, but including conservative amino acid substitutions in addition to identical amino acids in the computation.
  • a conservative amino acid substitution is one in which an amino acid is substituted for another amino acid having similar properties such that the folding or activity of the protein is not significantly affected.
  • Aromatic amino acids that can be substituted for each other are phenylalanine, tryptophan, and tyrosine; interchangeable hydrophobic amino acids are leucine, isoleucine, methionine, and valine; interchangeable polar amino acids are glutamine and asparagine; interchangeable basic amino acids are arginine, lysine and histidine; interchangeable acidic amino acids are aspartic acid and glutamic acid; and interchangeable small amino acids are alanine, serine, threonine, cysteine and glycine.
  • nucleic acid sequences are provided by the local homology algorithm of Smith and Waterman (Smith and Waterman, 1981, Advances in Applied Mathematics 2:482-489; database: European Bioinformatics Institute http://www.ebi.ac.uk/MPsrch/; Smith and Waterman, 1981, J. of Molec.Biol., 147:195-197; Nicholas et al., 1998, “A tutorial on Searching Sequence Databases and Sequence Scoring Methods” (www.psc.edu) and references cited therein.; W. R. Pearson, 1991, Genomics 11:635-650).
  • This algorithm can be applied to amino acid sequences by using the scoring matrix developed by Dayhoff (Dayhoff: Atlas of Protein Sequences and Structure, M. O. Dayhoff ed., 5 suppl. 3:353-358, National Biomedical Research Foundation, Washington, D.C., USA), and normalized by Gribskov (Gribskov 1986 Nucl. Acids Res. 14(6):6745-6763).
  • the Smith-Waterman algorithm may be employed where default parameters are used for scoring (for example, gap open penalty of 12, gap extension penalty of two). From the data generated, the “Match” value reflects “sequence identity.”
  • Derivative nucleic acid molecules of the subject nucleic acid molecules include sequences that hybridize to the nucleic acid sequence of any of SEQ ID NOs: 1, 2, 3, 4, or 5.
  • the stringency of hybridization can be controlled by temperature, ionic strength, pH, and the presence of denaturing agents such as formamide during hybridization and washing. Conditions routinely used are set out in readily available procedure texts (e.g., Current Protocol in Molecular Biology, Vol. 1, Chap. 2.10, John Wiley & Sons, Publishers (1994); Sambrook et al., Molecular Cloning, Cold Spring Harbor (1989)).
  • a nucleic acid molecule of the invention is capable of hybridizing to a nucleic acid molecule containing the nucleotide sequence of any one of SEQ ID NOs: 1, ,2 3, 4, or 5 under stringent hybridization conditions that comprise: prehybridization of filters containing nucleic acid for 8 hours to overnight at 65° C. in a solution comprising 6 ⁇ single strength citrate (SSC) (1 ⁇ SSC is 0.15 M NaCl, 0.015 M Na citrate; pH 7.0), 5 ⁇ Denhardt's solution, 0.05% sodium pyrophosphate and 100 ⁇ g/ml herring sperm DNA; hybridization for 18-20 hours at 65° C.
  • SSC single strength citrate
  • moderately stringent hybridization conditions comprise: pretreatment of filters containing nucleic acid for 6 h at 40° C. in a solution containing 35% formamide, 5 ⁇ SSC, 50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 0.1% PVP, 0.1% Ficoll, 1% BSA, and 500 ⁇ g/ml denatured salmon sperm DNA; hybridization for 18-20h at 40° C.
  • low stringency conditions can be used that comprise: incubation for 8 hours to overnight at 37° C. in a solution comprising 20% formamide, 5 ⁇ SSC, 50 mM sodium phosphate (pH 7.6), 5 ⁇ Denhardt's solution, 10% dextran sulfate, and 20 ⁇ g/ml denatured sheared salmon sperm DNA; hybridization in the same buffer for 18 to 20 hours; and washing of filters in 1 ⁇ SSC at about 37° C. for 1 hour.
  • HS2ST nucleic acids and polypeptides useful for identifying and testing agents that modulate HS2ST function and for other applications related to the involvement of HS2ST in the p53 pathway.
  • HS2ST nucleic acids and derivatives and orthologs thereof may be obtained using any available method. For instance, techniques for isolating cDNA or genomic DNA sequences of interest by screening DNA libraries or by using polymerase chain reaction (PCR) are well known in the art.
  • PCR polymerase chain reaction
  • the particular use for the protein will dictate the particulars of expression, production, and purification methods. For instance, production of proteins for use in screening for modulating agents may require methods that preserve specific biological activities of these proteins, whereas production of proteins for antibody generation may require structural integrity of particular epitopes.
  • HS2ST protein for assays used to assess HS2ST function, such as involvement in cell cycle regulation or hypoxic response, may require expression in eukaryotic cell lines capable of these cellular activities.
  • recombinant HS2ST is expressed in a cell line known to have defective p53 function (e.g. SAOS-2 osteoblasts, H1299 lung cancer cells, C33A and HT3 cervical cancer cells, HT-29 and DLD-1 colon cancer cells, among others, available from American Type Culture Collection (ATCC), Manassas, Va.).
  • the recombinant cells are used in cell-based screening assay systems of the invention, as described further below.
  • the nucleotide sequence encoding an HS2ST polypeptide can be inserted into any appropriate expression vector.
  • the necessary transcriptional and translational signals can derive from the native HS2ST gene and/or its flanking regions or can be heterologous.
  • a variety of host-vector expression systems may be utilized, such as mammalian cell systems infected with virus (e.g. vaccinia virus, adenovirus, etc.); insect cell systems infected with virus (e.g. baculovirus); microorganisms such as yeast containing yeast vectors, or bacteria transformed with bacteriophage, plasmid, or cosmid DNA.
  • a host cell strain that modulates the expression of, modifies, and/or specifically processes the gene product may be used.
  • the expression vector can comprise a promoter operably linked to an HS2ST gene nucleic acid, one or more origins of replication, and, one or more selectable markers (e.g. thymidine kinase activity, resistance to antibiotics, etc.).
  • selectable markers e.g. thymidine kinase activity, resistance to antibiotics, etc.
  • recombinant expression vectors can be identified by assaying for the expression of the HS2ST gene product based on the physical or functional properties of the HS2ST protein in in vitro assay systems (e.g. immunoassays).
  • the HS2ST protein, fragment, or derivative may be optionally expressed as a fusion, or chimeric protein product (i.e. it is joined via a peptide bond to a heterologous protein sequence of a different protein), for example to facilitate purification or detection.
  • a chimeric product can be made by ligating the appropriate nucleic acid sequences encoding the desired amino acid sequences to each other using standard methods and expressing the chimeric product.
  • a chimeric product may also be made by protein synthetic techniques, e.g. by use of a peptide synthesizer (Hunkapiller et al., Nature (1984) 310:105-111).
  • the gene product can be isolated and purified using standard methods (e.g. ion exchange, affinity, and gel exclusion chromatography; centrifugation; differential solubility; electrophoresis, cite purification reference).
  • native HS2ST proteins can be purified from natural sources, by standard methods (e.g. immunoaffinity purification).
  • a protein Once a protein is obtained, it may be quantified and its activity measured by appropriate methods, such as immunoassay, bioassay, or other measurements of physical properties, such as crystallography.
  • the methods of this invention may also use cells that have been engineered for altered expression (mis-expression) of HS2ST or other genes associated with the p53 pathway.
  • mis-expression encompasses ectopic expression, over-expression, under-expression, and non-expression (e.g. by gene knock-out or blocking expression that would otherwise normally occur).
  • Animal models that have been genetically modified to alter HS2ST expression may be used in in vivo assays to test for activity of a candidate p53 modulating agent, or to further assess the role of HS2ST in a p53 pathway process such as apoptosis or cell proliferation.
  • the altered HS2ST expression results in a detectable phenotype, such as decreased or increased levels of cell proliferation, angiogenesis, or apoptosis compared to control animals having normal HS2ST expression.
  • the genetically modified animal may additionally have altered p53 expression (e.g. p53 knockout).
  • Preferred genetically modified animals are mammals such as primates, rodents (preferably mice), cows, horses, goats, sheep, pigs, dogs and cats.
  • Preferred non-mammalian species include zebrafish, C. elegans, and Drosophila.
  • Preferred genetically modified animals are transgenic animals having a heterologous nucleic acid sequence present as an extrachromosomal element in a portion of its cells, i.e. mosaic animals (see, for example, techniques described by Jakobovits, 1994, Curr. Biol. 4:761-763.) or stably integrated into its germ line DNA (i.e., in the genomic sequence of most or all of its cells).
  • Heterologous nucleic acid is introduced into the germ line of such transgenic animals by genetic manipulation of, for example, embryos or embryonic stem cells of the host animal.
  • transgenic mice see Brinster et al., Proc. Nat. Acad. Sci. USA 82: 4438-4442 (1985), U.S. Pat. Nos. 4,736,866 and 4,870,009, both by Leder et al., U.S. Pat. No. 4,873,191 by Wagner et al., and Hogan, B., Manipulating the Mouse Embryo, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., (1986); for particle bombardment see U.S. Pat.
  • Clones of the nonhuman transgenic animals can be produced according to available methods (see Wilmut, I. et al. Nature 385:810-813; and PCT International Publication Nos. WO 97/07668 and WO 97/07669).
  • the transgenic animal is a “knock-out” animal having a heterozygous or homozygous alteration in the sequence of an endogenous HS2ST gene that results in a decrease of HS2ST function, preferably such that HS2ST expression is undetectable or insignificant.
  • Knock-out animals are typically generated by homologous recombination with a vector comprising a transgene having at least a portion of the gene to'be knocked out. Typically a deletion, addition or substitution has been introduced into the transgene to functionally disrupt it.
  • the transgene can be a human gene (e.g., from a human genomic clone) but more preferably is an ortholog of the human gene derived from the transgenic host species.
  • a mouse HS2ST gene is used to construct a homologous recombination vector suitable for altering an endogenous HS2ST gene in the mouse genome.
  • homologous recombination in mice are available (see Capecchi, Science (1989) 244:1288-1292; Joyner et al., Nature (1989) 338:153-156).
  • knock-out animals such as mice harboring a knockout of a specific gene, may be used to produce antibodies against the human counterpart of the gene that has been knocked out (Claesson M H et al., (1994) Scan J Immunol 40:257-264; Declerck P J et al., (1995) J Biol Chem. 270:8397-400).
  • the transgenic animal is a “knock-in” animal having an alteration in its genome that results in altered expression (e.g., increased (including ectopic) or decreased expression) of the HS2ST gene, e.g., by introduction of additional copies of HS2ST, or by operatively inserting a regulatory sequence that provides for altered expression of an endogenous copy of the HS2ST gene.
  • a regulatory sequence include inducible, tissue-specific, and constitutive promoters and enhancer elements.
  • the knock-in can be homozygous or heterozygous.
  • Transgenic nonhuman animals can also be produced that contain selected systems allowing for regulated expression of the transgene.
  • a system that may be produced is the cre/loxP recombinase system of bacteriophage P1 (Lakso et al., PNAS (1992) 89:6232-6236; U.S. Pat. No. 4,959,317). If a cre/loxP recombinase system is used to regulate expression of the transgene, animals containing transgenes encoding both the Cre recombinase and a selected protein are required.
  • Such animals can be provided through the construction of “double” transgenic animals, e.g., by mating two transgenic animals, one containing a transgene encoding a selected protein and the other containing a transgene encoding a recombinase.
  • a recombinase system is the FLP recombinase system of Saccharomyces cerevisiae (O'Gorman et al. (1991) Science 251:1351-1355; U.S. Pat. No. 5,654,182).
  • both Cre-LoxP and Flp-Frt are used in the same system to regulate expression of the transgene, and for sequential deletion of vector sequences in the same cell (Sun X et al (2000) Nat Genet 25:83-6).
  • the genetically modified animals can be used in genetic studies to further elucidate the p53 pathway, as animal models of disease and disorders implicating defective p53 function, and for in vivo testing of candidate therapeutic agents, such as those identified in screens described below.
  • the candidate therapeutic agents are administered to a genetically modified animal having altered HS2ST function and phenotypic changes are compared with appropriate control animals such as genetically modified animals that receive placebo treatment, and/or animals with unaltered HS2ST expression that receive candidate therapeutic agent.
  • animal models having defective p53 function can be used in the methods of the present invention.
  • a p53 knockout mouse can be used to assess, in vivo, the activity of a candidate p53 modulating agent identified in one of the in vitro assays described below.
  • p53 knockout mice are described in the literature (Jacks et al., Nature 2001;410:1111-1116, 1043-1044; Donehower et al., supra).
  • the candidate p53 modulating agent when administered to a model system with cells defective in p53 function, produces a detectable phenotypic change in the model system indicating that the p53 function is restored, i.e., the cells exhibit normal cell cycle progression.
  • the invention provides methods to identify agents that interact with and/or modulate the function of HS2ST and/or the p53 pathway. Such agents are useful in a variety of diagnostic and therapeutic applications associated with the p53 pathway, as well as in further analysis of the HS2ST protein and its contribution to the p53 pathway. Accordingly, the invention also provides methods for modulating the p53 pathway comprising the step of specifically modulating HS2ST activity by administering a HS2ST-interacting or -modulating agent.
  • HS2ST-modulating agents inhibit or enhance HS2ST activity or otherwise affect normal HS2ST function, including transcription, protein expression, protein localization, and cellular or extra-cellular activity.
  • the candidate p53 pathway- modulating agent specifically modulates the function of the HS2ST.
  • the phrases “specific modulating agent”, “specifically modulates”, etc., are used herein to refer to modulating agents that directly bind to the HS2ST polypeptide or nucleic acid, and preferably inhibit, enhance, or otherwise alter, the function of the HS2ST.
  • the term also encompasses modulating agents that alter the interaction of the HS2ST with a binding partner or substrate (e.g. by binding to a binding partner of an HS2ST, or to a protein/binding partner complex, and inhibiting function).
  • HS2ST-modulating agents include small molecule compounds; HS2ST-interacting proteins, including antibodies and other biotherapeutics; and nucleic acid modulators such as antisense and RNA inhibitors.
  • the modulating agents may be formulated in pharmaceutical compositions, for example, as compositions that may comprise other active ingredients, as in combination therapy, and/or suitable carriers or excipients. Techniques for formulation and administration of the compounds may be found in “Remington's Pharmaceutical Sciences” Mack Publishing Co., Easton, Pa., 19 th edition.
  • Small molecules are often preferred to modulate function of proteins with enzymatic function, and/or containing protein interaction domains.
  • Chemical agents referred to in the art as “small molecule” compounds are typically organic, non-peptide molecules, having a molecular weight less than 10,000, preferably less than 5,000, more preferably less than 1,000, and most preferably less than 500.
  • This class of modulators includes chemically synthesized molecules, for instance, compounds from combinatorial chemical libraries. Synthetic compounds may be rationally designed or identified based on known or inferred properties of the HS2ST protein or may be identified by screening compound libraries.
  • modulators of this class are natural products, particularly secondary metabolites from organisms such as plants or fungi, which can also be identified by screening compound libraries for HS2ST-modulating activity. Methods for generating and obtaining compounds are well known in the art (Schreiber SL, Science (2000) 151: 1964-1969; Radmann J and Gunther J, Science (2000)151:1947-1948).
  • Small molecule modulators identified from screening assays can be used as lead compounds from which candidate clinical compounds may be designed, optimized, and synthesized. Such clinical compounds may have utility in treating pathologies associated with the p53 pathway.
  • the activity of candidate small molecule modulating agents may be improved several-fold through iterative secondary functional validation, as further described below, structure determination, and candidate modulator modification and testing.
  • candidate clinical compounds are generated with specific regard to clinical and pharmacological properties.
  • the reagents may be derivatized and re-screened using in vitro and in vivo assays to optimize activity and minimize toxicity for pharmaceutical development.
  • HS2ST-interacting proteins are useful in a variety of diagnostic and therapeutic applications related to the p53 pathway and related disorders, as well as in validation assays for other HS2ST-modulating agents.
  • HS2ST-interacting proteins affect normal HS2ST function, including transcription, protein expression, protein localization, and cellular or extra-cellular activity.
  • HS2ST-interacting proteins are useful in detecting and providing information about the function of HS2ST proteins, as is relevant to p53 related disorders, such as cancer (e.g., for diagnostic means).
  • An HS2ST-interacting protein may be endogenous, i.e. one that naturally interacts genetically or biochemically with an HS2ST, such as a member of the HS2ST pathway that modulates HS2ST expression, localization, and/or activity.
  • HS2ST-modulators include dominant negative forms of HS2ST-interacting proteins and of HS2ST proteins themselves.
  • Yeast two-hybrid and variant screens offer preferred methods for identifying endogenous HS2ST-interacting proteins (Finley, R. L. et al. (1996) in DNA Cloning-Expression Systems: A Practical Approach, eds. Glover D. & Hames B. D (Oxford University Press, Oxford, England), pp. 169-203; Fashema SF et al., Gene
  • Mass spectrometry is an alternative preferred method for the elucidation of protein complexes (reviewed in, e.g., Pandley A and Mann M, Nature (2000) 405:837-846; Yates J R 3 rd , Trends Genet (2000) 16:5-8).
  • An HS2ST-interacting protein may be an exogenous protein, such as an HS2ST-specific antibody or a T-cell antigen receptor (see, e.g., Harlow and Lane (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory; Harlow and Lane (1999) Using antibodies: a laboratory manual. Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press).
  • HS2ST antibodies are further discussed below.
  • an HS2ST-interacting protein specifically binds an HS2ST protein.
  • an HS2ST-modulating agent binds an HS2ST substrate, binding partner, or cofactor.
  • the protein modulator is an HS2ST specific antibody agonist or antagonist.
  • the antibodies have therapeutic and diagnostic utilities, and can be used in screening assays to identify HS2ST modulators.
  • the antibodies can also be used in dissecting the portions of the HS2ST pathway responsible for various cellular responses and in the general processing and maturation of the HS2ST.
  • Antibodies that specifically bind HS2ST polypeptides can be generated using known methods.
  • the antibody is specific to a mammalian ortholog of HS2ST polypeptide, and more preferably, to human HS2ST.
  • Antibodies may be polyclonal, monoclonal (mAbs), humanized or chimeric antibodies, single chain antibodies, Fab fragments, F(ab′).sub.2 fragments, fragments produced by a FAb expression library, anti-idiotypic (anti-Id) antibodies, and epitope-binding fragments of any of the above.
  • Epitopes of HS2ST which are particularly antigenic can be selected, for example, by routine screening of HS2ST polypeptides for antigenicity or by applying a theoretical method for selecting antigenic regions of a protein (Hopp and Wood (1981), Proc. Nati. Acad. Sci. U.S.A. 78:3824-28; Hopp and Wood, (1983) Mol. Immunol. 20:483-89; Sutcliffe et al., (1983) Science 219:660-66) to the amino acid sequence shown in any of SEQ ID NOs:6, ,7 8, or 9.
  • Monoclonal antibodies with affinities of 10 8 M ⁇ 1 preferably 10 9 M ⁇ 1 to 10 10 M ⁇ 1 , or stronger can be made by standard procedures as described (Harlow and Lane, supra; Goding (1986) Monoclonal Antibodies: Principles and Practice (2d ed) Academic Press, New York; and U.S. Pat. Nos. 4,381,292; 4,451,570; and 4,618,577).
  • Antibodies may be generated against crude cell extracts of HS2ST or substantially purified fragments thereof. If HS2ST fragments are used, they preferably comprise at least 10, and more preferably, at least 20 contiguous amino acids of an HS2ST protein.
  • HS2ST-specific antigens and/or immunogens are coupled to carrier proteins that stimulate the immune response.
  • the subject polypeptides are covalently coupled to the keyhole limpet hemocyanin (KLH) carrier, and the conjugate is emulsified in Freund's complete adjuvant, which enhances the immune response.
  • KLH keyhole limpet hemocyanin
  • An appropriate immune system such as a laboratory rabbit or mouse is immunized according to conventional protocols.
  • HS2ST-specific antibodies is assayed by an appropriate assay such as a solid phase enzyme-linked immunosorbant assay (ELISA) using immobilized corresponding HS2ST polypeptides.
  • an appropriate assay such as a solid phase enzyme-linked immunosorbant assay (ELISA) using immobilized corresponding HS2ST polypeptides.
  • ELISA enzyme-linked immunosorbant assay
  • Other assays such as radioimmunoassays or fluorescent assays might also be used.
  • Chimeric antibodies specific to HS2ST polypeptides can be made that contain different portions from different animal species.
  • a human immunoglobulin constant region may be linked to a variable region of a murine mAb, such that the antibody derives its biological activity from the human antibody, and its binding specificity from the murine fragment.
  • Chimeric antibodies are produced by splicing together genes that encode the appropriate regions from each species (Morrison et al., Proc. Natl. Acad. Sci. (1984) 81:6851-6855; Neuberger et al., Nature (1984) 312:604-608; Takeda et al., Nature (1985) 31:452-454).
  • Humanized antibodies which are a form of chimeric antibodies, can be generated by grafting complementary-determining regions (CDRs) (Carlos, T. M., J. M. Harlan. 1994. Blood 84:2068-2101) of mouse antibodies into a background of human framework regions and constant regions by recombinant DNA technology (Riechmann L M, et al., 1988 Nature 323: 323-327). Humanized antibodies contain ⁇ 10% murine sequences and ⁇ 90% human sequences, and thus further reduce or eliminate immunogenicity, while retaining the antibody specificities (Co M S, and Queen C. 1991 Nature 351: 501-501; Morrison SL. 1992 Ann. Rev. Immun. 10:239-265). Humanized antibodies and methods of their production are well-known in the art (U.S. Pat. Nos. 5,530,101, 5,585,089, 5,693,762, and 6,180,370).
  • HS2ST-specific single chain antibodies which are recombinant, single chain polypeptides formed by linking the heavy and light chain fragments of the Fv regions via an amino acid bridge, can be produced by methods known in the art (U.S. Pat. No. 4,946,778; Bird, Science (1988) 242:423-426; Huston et al., Proc. Natl. Acad. Sci. USA
  • T-cell antigen receptors are included within the scope of antibody modulators (Harlow and Lane, 1988, supra).
  • polypeptides and antibodies of the present invention may be used with or without modification. Frequently, antibodies will be labeled by joining, either covalently or non-covalently, a substance that provides for a detectable signal, or that is toxic to cells that express the targeted protein (Menard S, et al., Int J. Biol Markers
  • labels and conjugation techniques include radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent moieties, fluorescent emitting lanthanide metals, chemiluminescent moieties, bioluminescent moieties, magnetic particles, and the like (U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; and 4,366,241). Also, recombinant immunoglobulins may be produced (U.S. Pat. No. 4,816,567). Antibodies to cytoplasmic polypeptides may be delivered and reach their targets by conjugation with membrane-penetrating toxin proteins (U.S. Pat. No. 6,086,900).
  • the antibodies of the subject invention are typically administered parenterally, when possible at the target site, or intravenously.
  • the therapeutically effective dose and dosage regimen is determined by clinical studies.
  • the amount of antibody administered is in the range of about 0.1 mg/kg—to about 10 mg/kg of patient weight.
  • the antibodies are formulated in a unit dosage injectable form (e.g., solution, suspension, emulsion) in association with a pharmaceutically acceptable vehicle.
  • a pharmaceutically acceptable vehicle are inherently nontoxic and non-therapeutic. Examples are water, saline, Ringer's solution, dextrose solution, and 5% human serum albumin.
  • Nonaqueous vehicles such as fixed oils, ethyl oleate, or liposome carriers may also be used.
  • the vehicle may contain minor amounts of additives, such as buffers and preservatives, which enhance isotonicity and chemical stability or otherwise enhance therapeutic potential.
  • the antibodies' concentrations in such vehicles are typically in the range of about 1 mg/ml to about 10 mg/ml. Immunotherapeutic methods are further described in the literature (U.S. Pat. No. 5,859,206; WO0073469).
  • HS2ST-modulating agents comprise nucleic acid molecules, such as antisense oligomers or double stranded RNA (dsRNA), which generally inhibit HS2ST activity.
  • Preferred nucleic acid modulators interfere with the function of the HS2ST nucleic acid such as DNA replication, transcription, translocation of the HS2ST RNA to the site of protein translation, translation of protein from the HS2ST RNA, splicing of the HS2ST RNA to yield one or more mRNA species, or catalytic activity which may be engaged in or facilitated by the HS2ST RNA.
  • the antisense oligomer is an oligonucleotide that is sufficiently complementary to an HS2ST mRNA to bind to and prevent translation, preferably by binding to the 5′ untranslated region.
  • HS2ST-specific antisense oligonucleotides preferably range from at least 6 to about 200 nucleotides.
  • the oligonucleotide is preferably at least 10, 15, or 20 nucleotides in length. In other embodiments, the oligonucleotide is preferably less than 50, 40, or 30 nucleotides in length.
  • the oligonucleotide can be DNA or RNA or a chimeric mixture or derivatives or modified versions thereof, single-stranded or double-stranded.
  • the oligonucleotide can be modified at the base moiety, sugar moiety, or phosphate backbone.
  • the oligonucleotide may include other appending groups such as peptides, agents that facilitate transport across the cell membrane, hybridization-triggered cleavage agents, and intercalating agents.
  • the antisense oligomer is a phosphothioate morpholino oligomer (PMO).
  • PMOs are assembled from four different morpholino subunits, each of which contain one of four genetic bases (A, C, G, or T) linked to a six-membered morpholine ring. Polymers of these subunits are joined by non-ionic phosphodiamidate intersubunit linkages. Details of how to make and use PMOs and other antisense oligomers are well known in the art (e.g. see WO99/18193; Probst J C, Antisense Oligodeoxynucleotide and Ribozyme Design, Methods. (2000) 22(3):271-281; Summerton J, and Weller D. 1997 Antisense Nucleic Acid Drug Dev.:7:187-95; U.S. Pat. Nos. 5,235,033; and 5,378,841).
  • RNAi is the process of sequence-specific, post-transcriptional gene silencing in animals and plants, initiated by double-stranded RNA (dsRNA) that is homologous in sequence to the silenced gene.
  • dsRNA double-stranded RNA
  • Methods relating to the use of RNAi to silence genes in C. elegans, Drosophila, plants, and humans are known in the art (Fire A, et al., 1998 Nature 391:806-811; Fire, A. Trends Genet. 15, 358-363 (1999); Sharp, P. A. RNA interference 2001. Genes Dev. 15, 485-490 (2001); Hammond, S.
  • Nucleic acid modulators are commonly used as research reagents, diagnostics, and therapeutics. For example, antisense oligonucleotides, which are able to inhibit gene expression with extraordinar specificity, are often used to elucidate the function of particular genes (see, for example, U.S. Pat. No. 6,165,790). Nucleic acid modulators are also used, for example, to distinguish between functions of various members of a biological pathway. For example, antisense oligomers have been employed as therapeutic moieties in the treatment of disease states in animals and man and have been demonstrated in numerous clinical trials to be safe and effective (Milligan J F, et al, Current Concepts in Antisense Drug Design, J Med Chem.
  • an HS2ST-specific nucleic acid modulator is used in an assay to further elucidate the role of the HS2ST in the p53 pathway, and/or its relationship to other members of the pathway.
  • an HS2ST-specific antisense oligomer is used as a therapeutic agent for treatment of p53-related disease states.
  • the invention provides assay systems and screening methods for identifying specific modulators of HS2ST activity.
  • an “assay system” encompasses all the components required for performing and analyzing results of an assay that detects and/or measures a particular event.
  • primary assays are used to identify or confirm a modulator's specific biochemical or molecular effect with respect to the HS2ST nucleic acid or protein.
  • secondary assays further assess the activity of a HS2ST modulating agent identified by a primary assay and may confirm that the modulating agent affects HS2ST in a manner relevant to the p53 pathway. In some cases, HS2ST modulators will be directly tested in a secondary assay.
  • the screening method comprises contacting a suitable assay system comprising an HS2ST polypeptide with a candidate agent under conditions whereby, but for the presence of the agent, the system provides a reference activity (e.g. transferase activity), which is based on the particular molecular event the screening method detects.
  • a reference activity e.g. transferase activity
  • the type of modulator tested generally determines the type of primary assay.
  • screening assays are used to identify candidate modulators. Screening assays may be cell-based or may use a cell-free system that recreates or retains the relevant biochemical reaction of the target protein (reviewed in Sittampalam G S et al., Curr Opin Chem Biol (1997) 1:384-91 and accompanying references).
  • the term “cell-based” refers to assays using live cells, dead cells, or a particular cellular fraction, such as a membrane, endoplasmic reticulum, or mitochondrial fraction.
  • cell free encompasses assays using substantially purified protein (either endogenous or recombinantly produced), partially purified or crude cellular extracts.
  • Screening assays may detect a variety of molecular events, including protein-DNA interactions, protein-protein interactions (e.g., receptor-ligand binding), transcriptional activity (e.g., using a reporter gene), enzymatic activity (e.g., via a property of the substrate), activity of second messengers, immunogenicty and changes in cellular morphology or other cellular characteristics.
  • Appropriate screening assays may use a wide range of detection methods including fluorescent, radioactive, calorimetric, spectrophotometric, and amperometric methods, to provide a read-out for the particular molecular event detected.
  • Cell-based screening assays usually require systems for recombinant expression of HS2ST and any auxiliary proteins demanded by the particular assay. Appropriate methods for generating recombinant proteins produce sufficient quantities of proteins that retain their relevant biological activities and are of sufficient purity to optimize activity and assure assay reproducibility. Yeast two-hybrid and variant screens, and mass spectrometry provide preferred methods for determining protein-protein interactions and elucidation of protein complexes. In certain applications, when HS2ST-interacting proteins are used in screens to identify small molecule modulators, the binding specificity of the interacting protein to the HS2ST protein may be assayed by various known methods such as substrate processing (e.g.
  • binding equilibrium constants usually at least about 10 7 M ⁇ 1 , preferably at least about 10 8 M ⁇ 1 , more preferably at least about 10 9 M ⁇ 1
  • immunogenicity e.g. ability to elicit HS2ST specific antibody in a heterologous host such as a mouse, rat, goat or rabbit.
  • binding may be assayed by, respectively, substrate and ligand processing.
  • the screening assay may measure a candidate agent's ability to specifically bind to or modulate activity of a HS2ST polypeptide, a fusion protein thereof, or to cells or membranes bearing the polypeptide or fusion protein.
  • the HS2ST polypeptide can be full length or a fragment thereof that retains functional HS2ST activity.
  • the HS2ST polypeptide may be fused to another polypeptide, such as a peptide tag for detection or anchoring, or to another tag.
  • the HS2ST polypeptide is preferably human HS2ST, or is an ortholog or derivative thereof as described above.
  • the screening assay detects candidate agent-based modulation of HS2ST interaction with a binding target, such as an endogenous or exogenous protein or other substrate that has HS2ST—specific binding activity, and can be used to assess normal HS2ST gene function.
  • a binding target such as an endogenous or exogenous protein or other substrate that has HS2ST—specific binding activity
  • screening assays are high throughput or ultra high throughput and thus provide automated, cost-effective means of screening compound libraries for lead compounds (Fernandes P B, Curr Opin Chem Biol (1998) 2:597-603; Sundberg S A, Curr Opin Biotechnol 2000, 11:47-53).
  • screening assays uses fluorescence technologies, including fluorescence polarization, time-resolved fluorescence, and fluorescence resonance energy transfer.
  • a variety of suitable assay systems may be used to identify candidate HS2ST and p53 pathway modulators (e.g. U.S. Pat. Nos. 5,550,019 and 6,133,437 (apoptosis assays); and U.S. Pat. No. 6,020,135 (p53 modulation), among others). Specific preferred assays are described in more detail below.
  • Assays for sulfotransferase activity are known in the art.
  • An example of a High throughput method is a continuous coupled enzyme assay for the spectrophotometric analysis of sulfotransferases using aryl sulfotransferase IV (Burkart M D, and Wong C H. (1999) Anal Biochem. 274:131-7).
  • This assay is based on the regeneration of 3′-phosphoadenosine-5′-phosphosulfate (PAPS) from the desulfated 3′-phosphoadenosine-5′-phosphate (PAP) by a recombinant aryl sulfotransferase using p-nitrophenyl sulfate as the sulfate donor and visible spectrophotometric indicator of enzyme turnover.
  • PAPS 3′-phosphoadenosine-5′-phosphosulfate
  • PAP desulfated 3′-phosphoadenosine-5′-phosphate
  • Assays for apoptosis may be performed by terminal deoxynucleotidyl transferase-mediated digoxigenin-11-dUTP nick end labeling (TUNEL) assay.
  • TUNEL assay is used to measure nuclear DNA fragmentation characteristic of apoptosis (Lazebnik et al., 1994, Nature 371, 346), by following the incorporation of fluorescein-dUTP (Yonehara et al., 1989, J. Exp. Med. 169, 1747).
  • Apoptosis may further be assayed by acridine orange staining of tissue culture cells (Lucas, R., et al., 1998, Blood 15:4730-41).
  • An apoptosis assay system may comprise a cell that expresses an HS2ST, and that optionally has defective p53 function (e.g. p53 is over-expressed or under-expressed relative to wild-type cells).
  • a test agent can be added to the apoptosis assay system and changes in induction of apoptosis relative to controls where no test agent is added, identify candidate p53 modulating agents.
  • an apoptosis assay may be used as a secondary assay to test a candidate p53 modulating agents that is initially identified using a cell-free assay system.
  • An apoptosis assay may also be used to test whether HS2ST function plays a direct role in apoptosis.
  • an apoptosis assay may be performed on cells that over- or under-express HS2ST relative to wild type cells. Differences in apoptotic response compared to wild type cells suggests that the HS2ST plays a direct role in the apoptotic response.
  • Apoptosis assays are described further in U.S. Pat. No. 6,133,437.
  • Cell proliferation may be assayed via bromodeoxyuridine (BRDU) incorporation.
  • BRDU bromodeoxyuridine
  • This assay identifies a cell population undergoing DNA synthesis by incorporation of BRDU into newly-synthesized DNA. Newly-synthesized DNA may then be detected using an anti-BRDU antibody (Hoshino et al., 1986, Int. J. Cancer 38, 369; Campana et al., 1988, J. Immunol. Meth. 107, 79), or by other means.
  • Cell Proliferation may also be examined using [ 3 H]-thymidine incorporation (Chen, J., 1996, Oncogene 13:1395-403; Jeoung, J., 1995, J. Biol. Chem. 270:18367-73).
  • This assay allows for quantitative characterization of S-phase DNA syntheses.
  • cells synthesizing DNA will incorporate [ 3 H]-thymidine into newly synthesized DNA.
  • Incorporation can then be measured by standard techniques such as by counting of radioisotope in a scintillation counter (e.g., Beckman LS 3800 Liquid Scintillation Counter).
  • Cell proliferation may also be assayed by colony formation in soft agar (Sambrook et al., Molecular Cloning, Cold Spring Harbor (1989)). For example, cells transformed with HS2ST are seeded in soft agar plates, and colonies are measured and counted after two weeks incubation.
  • Involvement of a gene in the cell cycle may be assayed by flow cytometry (Gray J W et al. (1986) Int J Radiat Biol Relat Stud Phys Chem Med 49:237-55). Cells transfected with an HS2ST may be stained with propidium iodide and evaluated in a flow cytometer (available from Becton Dickinson).
  • a cell proliferation or cell cycle assay system may comprise a cell that expresses an HS2ST, and that optionally has defective p53 function (e.g. p53 is over-expressed or under-expressed relative to wild-type cells).
  • a test agent can be added to the assay system and changes in cell proliferation or cell cycle relative to controls where no test agent is added, identify candidate p53 modulating agents.
  • the cell proliferation or cell cycle assay may be used as a secondary assay to test a candidate p53 modulating agents that is initially identified using another assay system such as a cell-free kinase assay system.
  • a cell proliferation assay may also be used to test whether HS2ST function plays a direct role in cell proliferation or cell cycle.
  • a cell proliferation or cell cycle assay may be performed on cells that over- or under-express HS2ST relative to wild type cells. Differences in proliferation or cell cycle compared to wild type cells suggests that the HS2ST plays a direct role in cell proliferation or cell cycle.
  • Angiogenesis may be assayed using various human endothelial cell systems, such as umbilical vein, coronary artery, or dermal cells. Suitable assays include Alamar Blue based assays (available from Biosource International) to measure proliferation; migration assays using fluorescent molecules, such as the use of Becton Dickinson Falcon HTS FluoroBlock cell culture inserts to measure migration of cells through membranes in presence or absence of angiogenesis enhancer or suppressors; and tubule formation assays based on the formation of tubular structures by endothelial cells on Matrigel® (Becton Dickinson).
  • Alamar Blue based assays available from Biosource International
  • migration assays using fluorescent molecules such as the use of Becton Dickinson Falcon HTS FluoroBlock cell culture inserts to measure migration of cells through membranes in presence or absence of angiogenesis enhancer or suppressors
  • tubule formation assays based on the formation of tubular structures by endothelial cells on Ma
  • an angiogenesis assay system may comprise a cell that expresses an HS2ST, and that optionally has defective p53 function (e.g. p53 is over-expressed or under-expressed relative to wild-type cells).
  • a test agent can be added to the angiogenesis assay system and changes in angiogenesis relative to controls where no test agent is added, identify candidate p53 modulating agents.
  • the angiogenesis assay may be used as a secondary assay to test a candidate p53 modulating agents that is initially identified using another assay system.
  • An angiogenesis assay may also be used to test whether HS2ST function plays a direct role in cell proliferation. For example, an angiogenesis assay may be performed on cells that over- or under-express HS2ST relative to wild type cells. Differences in angiogenesis compared to wild type cells suggests that the HS2ST plays a direct role in angiogenesis.
  • hypoxia inducible factor-1 The alpha subunit of the transcription factor, hypoxia inducible factor-1 (HIF-1), is upregulated in tumor cells following exposure to hypoxia in vitro.
  • HIF-1 hypoxia inducible factor-1
  • hypoxic conditions stimulates the expression of genes known to be important in tumour cell survival, such as those encoding glyolytic enzymes and VEGF.
  • Induction of such genes by hypoxic conditions may be assayed by growing cells transfected with HS2ST in hypoxic conditions (such as with 0.1% O2, 5% CO2, and balance N2, generated in a Napco 7001 incubator (Precision Scientific)) and normoxic conditions, followed by assessment of gene activity or expression by Taqman®.
  • a hypoxic induction assay system may comprise a cell that expresses an HS2ST, and that optionally has a mutated p53 (e.g. p53 is over-expressed or under-expressed relative to wild-type cells).
  • a test agent can be added to the hypoxic induction assay system and changes in hypoxic response relative to controls where no test agent is added, identify candidate p53 modulating agents.
  • the hypoxic induction assay may be used as a secondary assay to test a candidate p53 modulating agents that is initially identified using another assay system.
  • a hypoxic induction assay may also be used to test whether HS2ST function plays a direct role in the hypoxic response.
  • hypoxic induction assay may be performed on cells that over- or under-express HS2ST relative to wild type cells. Differences in hypoxic response compared to wild type cells suggests that the HS2ST plays a direct role in hypoxic induction.
  • Cell adhesion assays measure adhesion of cells to purified adhesion proteins, or adhesion of cells to each other, in presence or absence of candidate modulating agents.
  • Cell-protein adhesion assays measure the ability of agents to modulate the adhesion of cells to purified proteins. For example, recombinant proteins are produced, diluted to 2.5g/mL in PBS, and used to coat the wells of a microtiter plate. The wells used for negative control are not coated. Coated wells are then washed, blocked with 1% BSA, and washed again. Compounds are diluted to 2 ⁇ final test concentration and added to the blocked, coated wells. Cells are then added to the wells, and the unbound cells are washed off. Retained cells are labeled directly on the plate by adding a membrane-permeable fluorescent dye, such as calcein-AM, and the signal is quantified in a fluorescent microplate reader.
  • a membrane-permeable fluorescent dye such as calcein-AM
  • Cell-cell adhesion assays measure the ability of agents to modulate binding of cell adhesion proteins with their native ligands. These assays use cells that naturally or recombinantly express the adhesion protein of choice.
  • cells expressing the cell adhesion protein are plated in wells of a multiwell plate.
  • Cells expressing the ligand are labeled with a membrane-permeable fluorescent dye, such as BCECF, and allowed to adhere to the monolayers in the presence of candidate agents. Unbound cells are washed off, and bound cells are detected using a fluorescence plate reader.
  • High-throughput cell adhesion assays have also been described.
  • small molecule ligands and peptides are bound to the surface of microscope slides using a microarray spotter, intact cells are then contacted with the slides, and unbound cells are washed off.
  • this assay not only the binding specificity of the peptides and modulators against cell lines are determined, but also the functional cell signaling of attached cells using immunofluorescence techniques in situ on the microchip is measured (Falsey J R et al., Bioconjug Chem. May-June 2001;12(3):346-53).
  • HS2ST-specific antibodies For antibody modulators, appropriate primary assays test is a binding assay that tests the antibody's affinity to and specificity for the HS2ST protein. Methods for testing antibody affinity and specificity are well known in the art (Harlow and Lane, 1988, 1999, supra).
  • the enzyme-linked immunosorbant assay (ELISA) is a preferred method for detecting HS2ST-specific antibodies; others include FACS assays, radioimmunoassays, and fluorescent assays.
  • primary assays may test the ability of the nucleic acid modulator to inhibit or enhance HS2ST gene expression, preferably mRNA expression.
  • expression analysis comprises comparing HS2ST expression in like populations of cells (e.g., two pools of cells that endogenously or recombinantly express HS2ST) in the presence and absence of the nucleic acid modulator. Methods for analyzing mRNA and protein expression are well known in the art.
  • HS2ST mRNA expression may be confirmed in cells treated with the nucleic acid modulator (e.g., Current Protocols in Molecular Biology (1994) Ausubel F M et al., eds., John Wiley & Sons, Inc., chapter 4; Freeman W M et al., Biotechniques (1999) 26:112-125; Kallioniemi O P, Ann Med 2001, 33:142-147; Blohm D H and Guiseppi-Elie, A Curr Opin Biotechnol 2001, 12:41-47).
  • the nucleic acid modulator e.g., Current Protocols in Molecular Biology (1994) Ausubel F M et al., eds., John Wiley & Sons, Inc., chapter 4; Freeman W M et al., Biotechniques (1999) 26:112-125; Kallioniemi O P, Ann Med 2001, 33:142-147; Blohm D H and Guiseppi-
  • Protein expression may also be monitored. Proteins are most commonly detected with specific antibodies or antisera directed against either the HS2ST protein or specific peptides. A variety of means including Western blotting, ELISA, or in situ detection, are available (Harlow E and Lane D, 1988 and 1999, supra).
  • Secondary assays may be used to further assess the activity of HS2ST-modulating agent identified by any of the above methods to confirm that the modulating agent affects HS2ST in a manner relevant to the p53 pathway.
  • HS2ST-modulating agents encompass candidate clinical compounds or other agents derived from previously identified modulating agent. Secondary assays can also be used to test the activity of a modulating agent on a particular genetic or biochemical pathway or to test the specificity of the modulating agent's interaction with HS2ST.
  • Secondary assays generally compare like populations of cells or animals (e.g., two pools of cells or animals that endogenously or recombinantly express HS2ST) in the presence and absence of the candidate modulator. In general, such assays test whether treatment of cells or animals with a candidate HS2ST-modulating agent results in changes in the p53 pathway in comparison to untreated (or mock- or placebo-treated) cells or animals. Certain assays use “sensitized genetic backgrounds”, which, as used herein, describe cells or animals engineered for altered expression of genes in the p53 or interacting pathways.
  • Cell based assays may use a variety of mammalian cell lines known to have defective p53 function (e.g. SAOS-2 osteoblasts, H1299 lung cancer cells, C33A and HT3 cervical cancer cells, HT-29 and DLD-1 colon cancer cells, among others, available from American Type Culture Collection (ATCC), Manassas, Va.). Cell based assays may detect endogenous p53 pathway activity or may rely on recombinant expression of p53 pathway components. Any of the aforementioned assays may be used in this cell-based format.
  • Candidate modulators are typically added to the cell media but may also be injected into cells or delivered by any other efficacious means.
  • Models for defective p53 pathway typically use genetically modified animals that have been engineered to mis-express (e.g., over-express or lack expression in) genes involved in the p53 pathway.
  • Assays generally require systemic delivery of the candidate modulators, such as by oral administration, injection, etc.
  • p53 pathway activity is assessed by monitoring neovascularization and angiogenesis.
  • Animal models with defective and normal p53 are used to test the candidate modulator's affect on HS2ST in Matrigel® assays.
  • Matrigel® is an extract of basement membrane proteins, and is composed primarily of laminin, collagen IV, and heparin sulfate proteoglycan. It is provided as a sterile liquid at 4° C., but rapidly forms a solid gel at 37° C. Liquid Matrigel® is mixed with various angiogenic agents, such as bFGF and VEGF, or with human tumor cells which over-express the HS2ST.
  • mice Female athymic nude mice (Taconic, Germantown, N.Y.) to support an intense vascular response.
  • Mice with Matrigel® pellets may be dosed via oral (PO), intraperitoneal (IP), or intravenous (IV) routes with the candidate modulator. Mice are euthanized 5-12 days post-injection, and the Matrigel® pellet is harvested for hemoglobin analysis (Sigma plasma hemoglobin kit). Hemoglobin content of the gel is found to correlate the degree of neovascularization in the gel.
  • the effect of the candidate modulator on HS2ST is assessed via tumorigenicity assays.
  • xenograft human tumors are implanted SC into female athymic mice, 6-7 week old, as single cell suspensions either from a pre-existing tumor or from in vitro culture.
  • the tumors which express the HS2ST endogenously are injected in the flank, 1 ⁇ 10 5 to 1 ⁇ 10 7 cells per mouse in a volume of 100 ⁇ L using a 27gauge needle. Mice are then ear tagged and tumors are measured twice weekly.
  • Candidate modulator treatment is initiated on the day the mean tumor weight reaches 100 mg.
  • Candidate modulator is delivered IV, SC, IP, or PO by bolus administration.
  • dosing can be performed multiple times per day.
  • the tumor weight is assessed by measuring perpendicular diameters with a caliper and calculated by multiplying the measurements of diameters in two dimensions.
  • the excised tumors maybe utilized for biomarker identification or further analyses.
  • immunohistochemistry staining xenograft tumors are fixed in 4% paraformaldehyde, 0. IM phosphate, pH 7.2, for 6 hours at 4° C., immersed in 30% sucrose in PBS, and rapidly frozen in isopentane cooled with liquid nitrogen.
  • HS2ST-modulating agents are useful in a variety of diagnostic and therapeutic applications where disease or disease prognosis is related to defects in the p53 pathway, such as angiogenic, apoptotic, or cell proliferation disorders.
  • the invention also provides methods for modulating the p53 pathway in a cell, preferably a cell pre-determined to have defective p53 function, comprising the step of administering an agent to the cell that specifically modulates HS2ST activity.
  • the modulating agent produces a detectable phenotypic change in the cell indicating that the p53 function is restored, i.e., for example, the cell undergoes normal proliferation or progression through the cell cycle.
  • Various expression analysis methods can be used to diagnose whether HS2ST expression occurs in a particular sample, including Northern blotting, slot blotting, ribonuclease protection, quantitative RT-PCR, and microarray analysis.
  • Tissues having a disease or disorder implicating defective p53 signaling that express an HS2ST are identified as amenable to treatment with an HS2ST modulating agent.
  • the p53 defective tissue overexpresses an HS2ST relative to normal tissue.
  • a Northern blot analysis of mRNA from tumor and normal cell lines, or from tumor and matching normal tissue samples from the same patient, using full or partial HS2ST cDNA sequences as probes can determine whether particular tumors express or overexpress HS2ST.
  • the TaqMan® is used for quantitative RT-PCR analysis of HS2ST expression in cell lines, normal tissues and tumor samples (PE Applied Biosystems).
  • HS2ST oligonucleotides and antibodies directed against an HS2ST, as described above for: (1) the detection of the presence of HS2ST gene mutations, or the detection of either over- or under-expression of HS2ST mRNA relative to the non-disorder state; (2) the detection of either an over- or an under-abundance of HS2ST gene product relative to the non-disorder state; and (3) the detection of perturbations or abnormalities in the signal transduction pathway mediated by HS2ST.
  • reagents such as the HS2ST oligonucleotides, and antibodies directed against an HS2ST, as described above for: (1) the detection of the presence of HS2ST gene mutations, or the detection of either over- or under-expression of HS2ST mRNA relative to the non-disorder state; (2) the detection of either an over- or an under-abundance of HS2ST gene product relative to the non-disorder state; and (3) the detection of perturbations or abnormalities in the
  • the invention is drawn to a method for diagnosing a disease in a patient, the method comprising: a) obtaining a biological sample from the patient; b) contacting the sample with a probe for HS2ST expression; c) comparing results from step (b) with a control; and d) determining whether step (c) indicates a likelihood of disease.
  • the disease is cancer, most preferably a cancer as shown in TABLE 1.
  • the probe may be either DNA or protein, including an antibody.
  • the Drosophila p53 gene was overexpressed specifically in the wing using the vestigial margin quadrant enhancer.
  • Increasing quantities of Drosophila p53 (titrated using different strength transgenic inserts in 1 or 2 copies) caused deterioration of normal wing morphology from mild to strong, with phenotypes including disruption of pattern and polarity of wing hairs, shortening and thickening of wing veins, progressive crumpling of the wing and appearance of dark “death” inclusions in wing blade.
  • BLAST analysis (Altschul et al., supra) was employed to identify Targets from Drosophila modifiers.
  • HS2ST GI#6912420, SEQ ID NO:7
  • HS2ST GI#5032219, SEQ ID NO:9
  • TM-HMM Error L. L. Sonnhammer, Gunnar von Heijne, and Anders Krogh: A hidden Markov model for predicting transmembrane helices in protein sequences.
  • TM-HMM Error L. L. Sonnhammer, Gunnar von Heijne, and Anders Krogh: A hidden Markov model for predicting transmembrane helices in protein sequences.
  • clust Remm M, and Sonnhammer E. Classification of transmembrane protein families in the Caenorhabditis elegans genome and identification of human orthologs. Genome Res. November 2000; 10(11): 1679-89) programs.
  • Fluorescently-labeled HS2ST peptide/substrate are added to each well of a 96-well microtiter plate, along with a test agent in a test buffer (10 mM HEPES, 10 mM NaCl, 6 mM magnesium chloride, pH 7.6). Changes in fluorescence polarization, determined by using a Fluorolite FPM-2 Fluorescence Polarization Microtiter System (Dynatech Laboratories, Inc), relative to control values indicates the test compound is a candidate modifier of HS2ST activity.
  • 33 P-labeled HS2ST peptide is added in an assay buffer (100 mM KCl, 20 mM HEPES pH 7.6, 1 mM MgCl 2 , 1% glycerol, 0.5% NP-40, 50 mM beta-mercaptoethanol, 1 mg/ml BSA, cocktail of protease inhibitors) along with a test agent to the wells of a Neutralite-avidin coated assay plate and incubated at 25° C. for 1 hour. Biotinylated substrate is then added to each well and incubated for 1 hour. Reactions are stopped by washing with PBS, and counted in a scintillation counter. Test agents that cause a difference in activity relative to control without test agent are identified as candidate p53 modulating agents.
  • proteins bound to the beads are solubilized by boiling in SDS sample buffer, fractionated by SDS-polyacrylamide gel electrophoresis, transferred to polyvinylidene difluoride membrane and blotted with the indicated antibodies.
  • the reactive bands are visualized with horseradish peroxidase coupled to the appropriate secondary antibodies and the enhanced chemiluminescence (ECL) Western blotting detection system (Amersham Pharmacia Biotech).
  • TaqMan analysis was used to assess expression levels of the disclosed genes in various samples.
  • primer pairs were designed to span introns to eliminate genomic contamination
  • each primer pair produced only one product.
  • Taqman reactions were carried out following manufacturer's protocols, in 25 ⁇ l total volume for 96-well plates and 10 ⁇ l total volume for 384-well plates, using 300 nM primer and 250 nM probe, and approximately 25 ng of cDNA.
  • the standard curve for result analysis was prepared using a universal pool of human cDNA samples, which is a mixture of cDNAs from a wide variety of tissues so that the chance that a target will be present in appreciable amounts is good.
  • the raw data were normalized using 18S rRNA (universally expressed in all tissues and cells).
  • tumor tissue samples were compared with matched normal tissues from the same patient.
  • a gene was considered overexpressed in a tumor when the level of expression of the gene was 2 fold or higher in the tumor compared with its matched normal sample.
  • a universal pool of cDNA samples was used instead.
  • a gene was considered overexpressed in a tumor sample when the difference of expression levels between a tumor sample and the average of all normal samples from the same tissue type was greater than 2 times the standard deviation of all normal samples (i.e., Tumor—average(all normal samples)>2 ⁇ STDEV(all normal samples)).
  • Results are shown in Table 1. Data presented in bold indicate that greater than 50% of tested tumor samples of the tissue type indicated in row 1 exhibited over expression of the gene listed in column 1, relative to normal samples. Underlined data indicates that between 25% to 49% of tested tumor samples exhibited over expression.
  • a modulator identified by an assay described herein can be further validated for therapeutic effect by administration to a tumor in which the gene is overexpressed. A decrease in tumor growth confirms therapeutic utility of the modulator.
  • the likelihood that the patient will respond to treatment can be diagnosed by obtaining a tumor sample from the patient, and assaying for expression of the gene targeted by the modulator.
  • the expression data for the gene(s) can also be used as a diagnostic marker for disease progression.
  • the assay can be performed by expression analysis as described above, by antibody directed to the gene target, or by any other available detection method.
  • TABLE 1 breast colon lung ovary HS2ST 2 12 12 30 5 14 1 7 (SEQ ID NO: 3) GI #4803743 1 12 2 30 1 14 0 7 (SEQ ID NO: 5)

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US10/160,758 Abandoned US20030036076A1 (en) 2001-06-05 2002-06-03 CADs as modifiers of the p53 pathway and methods of use
US10/161,398 Abandoned US20030013144A1 (en) 2001-06-05 2002-06-03 HS2STs as modifiers of the p53 pathway and methods of use
US11/378,923 Abandoned US20060160764A1 (en) 2001-06-05 2006-03-17 LIMKs as modifiers of p53 pathway and methods of use
US11/627,976 Abandoned US20080166709A1 (en) 2001-06-05 2007-01-28 MARKs as Modifiers of the p53 Pathway and Methods of Use
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