WO2002072753A2 - Peptides de thymidylate synthase se fixant a l'arnm de la thymidylate synthase - Google Patents

Peptides de thymidylate synthase se fixant a l'arnm de la thymidylate synthase Download PDF

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WO2002072753A2
WO2002072753A2 PCT/US2002/006634 US0206634W WO02072753A2 WO 2002072753 A2 WO2002072753 A2 WO 2002072753A2 US 0206634 W US0206634 W US 0206634W WO 02072753 A2 WO02072753 A2 WO 02072753A2
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sεq
peptide
human
coli
thymidylate synthase
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PCT/US2002/006634
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WO2002072753A3 (fr
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Carmen J. Allegra
Donna M. Voeller
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The Government Of The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1003—Transferases (2.) transferring one-carbon groups (2.1)
    • C12N9/1007—Methyltransferases (general) (2.1.1.)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/164—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • This application relates to the field of thymidylate synthase, an enzyme involved in the sensitivity of cancer cells to therapeutic agents, and to peptides that inhibit the translation of mRNA encoding thymidylate synthase.
  • Thymidylate synthase represents a central enzyme in the metabolic pathway for the de novo synthesis of thymidylate.
  • TS is a folate- dependent enzyme that catalyzes the reductive methylation of 2'-deoxuridine- 5'monphosphate (dUMP) by the reduced folate-5,10-methylenetetrahydrofolte to thymidine 5 '-monophosphate (dTMP, thymidylate).
  • dUMP 2'-deoxuridine- 5'monphosphate
  • dTMP thymidylate
  • TS-catalyzed reaction is the sole intracellular de novo source of dTMP. Given its central role in the biosynthesis of dTMP, an essential DNA precursor, and given that inhibition of this reaction results in prompt cessation of cellular proliferation and growth, TS represents an important target for cancer chemotherapy.
  • TS is a homodimeric protein formed from identical subunits.
  • the enzyme varies in molecular weight between approximately 60 to 75 kDa, depending upon the particular species from which it was derived.
  • the enzyme has been purified and well-characterized from a number of species, including human, yeast, Escherichia coli (E. coli), bacteriophage T4, viruses, parasites, mouse, and rat.
  • the crystal structure of TS from several species has also been resolved. Careful analysis of the predicted primary amino acid sequence of TS isolated from eight different species reveals that it is one of the most highly conserved proteins identified.
  • the genomic and cDNA clones of a number of different sources of TS have now been isolated and characterized.
  • human and mouse cDNAs have been isolated, and a number of molecular probes for the human and mouse sequences have been generated.
  • Full-length human TS protein is known to bind human TS mRNA with a high affinity, and is known to bind other cellular RNA species.
  • the binding site on the TS mRNA for TS protein has been analyzed in detail (Chu and Allegra, BioEssays 18:191-198, 1995).
  • TS is a target for the development of chemotherapeutic agents (Grem, J.L. (1996) in Cancer Chemotherapy and Bio therapy Principles and Practice (Chabner, B.A., and Longo, D.L., Eds.) pp. 149-211, Lippincott-Raven Publishers,
  • nucleotide prodrug specifically target TS and have been shown to have broad clinical utility in the treatment of cancer (Grem, J.L.
  • TS levels can be lowered by the use of antisense molecules.
  • Treatment with antisense nucleic acids targeted to TS resulted in enhanced sensitivity to fluoropyrimidines (Ju et al., Clin.Cancer Res. 4, 2229-2236, 1998).
  • the intracellular level of TS in the malignant tissue of patients may be used to predict response to fluoropyrimidine-based therapies (Lenz et al., Clin. Cancer Res. 4, 1243- 1250, 1998; Leichman et al., J. Clin. Oncol. 15, 3223-3229, 1997).
  • TS peptides which bind to TS mRNA. These peptides can be used to inhibit the translation of TS mRNA, and therefore are of use in screening assays to identify agents that bind TS mRNA, or that inhibit the binding of TS protein to TS mRNA. These peptides are also of use in treating subjects in conjunction with other chemotherapeutic agents, and in identifying molecules and mimetics that bind TS mRNA or bind the bimolecular complex of TS protein and TS mRNA. In one embodiment, a peptide is disclosed that consists essentially of at least
  • a method for inhibiting the translation of a mRNA encoding thymidylate synthase.
  • the method includes contacting the mRNA, or a cell expressing the mRNA, with a peptide consisting essentially of from about 10 consecutive amino acids to about 25 amino acids of the peptide interface region of thymidylate synthase.
  • the peptide binds to the mRNA encoding thymidylate synthase and inhibits the translation of the mRNA encoding thymidylate synthase.
  • a method for enhancing a chemotherapeutic effect of a chemotherapeutic agent in a cell contacted with the chemotherapeutic agent.
  • the method includes contacting the cell with a peptide consisting essentially of from about 10 consecutive amino acids to about 25 amino acids of the peptide interface region of thymidylate synthase.
  • the peptide binds to a mRNA encoding thymidylate synthase, and inhibits the translation of the mRNA, thereby enhancing the chemotherapeutic effect of the chemotherapeutic agent.
  • a method for treating a subject with a neoplastic disorder.
  • the method includes administering to the subject a chemotherapeutic agent and a thymidylate synthase peptide consisting essentially of a peptide from about 10 consecutive amino acids to about 25 amino acids of the peptide interface region of thymidylate synthase.
  • the peptide binds to the mRNA encoding thymidylate synthase, thereby inhibiting the translation of the mRNA and treating the neoplastic disorder.
  • the chemotherapeutic agent is an agent that targets TS, such as a nucleotide prodrug or an antifolate drug.
  • a pharmaceutical composition includes a thymidylate synthase peptide consisting essentially of from about 10 consecutive amino acids of to about 25 amino acids of the peptide interface region of thymidylate synthase, wherein the peptide binds to an mRNA encoding thymidylate synthase, and a pharmaceutically acceptable carrier.
  • a method for identifying an agent that binds a mRNA encoding thymidylate synthase.
  • the method includes contacting the mRNA encoding thymidylate synthase, or a cell in vitro comprising the mRNA encoding thymidylate synthase, with a thymidylate synthase peptide consisting essentially of from about 10 consecutive amino acids to about 25 amino acids of the peptide interface region of thymidylate synthase, wherein the peptide binds to the mRNA encoding thymidylate synthase and with the agent.
  • the method also includes evaluating the binding of the agent to the mRNA encoding thymidylate synthase.
  • a method for treating a subject with a neoplastic disorder includes administering to the subject a chemotherapeutic agent and a therapeutic nucleic acid sequence comprising a promoter operably linked to a nucleic acid sequence encoding a thymidylate synthase peptide consisting essentially of from about 10 consecutive amino acids to about 25 amino acids of the peptide interface region of thymidylate synthase.
  • the thymidylate synthase peptide binds to the mRNA encoding thymidylate synthase, thereby inhibiting the translation of the mRNA encoding thymidylate synthase, and thereby treating the subject.
  • Fig. 1 is a set of schematic diagrams showing the binding of TS peptides to TS mRNA.
  • Fig.lA is a ribbon structure diagram of the monomeric subunit of E. coli thymidylate synthase (TS) protein with each of the human binding peptides superimposed (peptide 9; peptide 14; peptide 29; peptide 38; peptide 43) and shown in shading. For orientation, the nucleotide substrate dUMP is illustrated in the active site of the enzyme.
  • Fig. IB is a diagram which represents the sequence of the full-length human thymidylate synthase protein with the binding peptides bracketed and in bold.
  • Fig. 2 shows the nucleic acid sequence (SEQ ID NO: 13) of human TS mRNA, and the amino acid sequence (SEQ ID NO: 14) of human thymidylate synthase protein.
  • chemotherapeutic agent is an agent, such as an antibody, chemical compound, molecule, peptidomimetic, or protein used for the treatment of a neoplastic disorder.
  • cDNA complementary DNA: A piece of DNA lacking internal, non- coding segments (introns) and regulatory sequences that determine transcription. cDNA is synthesized in the laboratory by reverse transcription from messenger RNA extracted from cells.
  • a conservative substitution is an amino acid substitution that does not affect the charge, hydrophobicity, or function of a protein or peptide.
  • a conservative substitution is an amino acid substitution in a TS peptide that does not substantially affect the ability of the peptide to bind TS mRNA.
  • Val He; Leu Degenerate variant A polynucleotide encoding a TS peptide that binds TS mRNA that includes a sequence that is degenerate as a result of the genetic code. There are 20 natural amino acids, most of which are specified by more than one codon. Therefore, all degenerate nucleotide sequences are included in the invention as long as the amino acid sequence of the TS peptide encoded by the nucleotide sequence is unchanged.
  • Expression Control Sequences Nucleic acid sequences that regulate the expression of a nucleic acid sequence to which it is operatively linked. Expression control sequences are operatively linked to a nucleic acid sequence when the expression control sequences control and regulate the transcription and, as appropriate, translation of the nucleic acid sequence.
  • expression control sequences can include appropriate promoters, enhancers, transcription terminators, a start codon (i.e., ATG) in front of a protein-encoding gene, splicing signal for ulcerrons, maintenance of the correct reading frame of that gene to permit proper translation of mRNA, and stop codons.
  • control sequences is intended to include, at a minimum, components whose presence can influence expression, and can also include additional components whose presence is advantageous, for example, leader sequences and fusion partner sequences.
  • Expression control sequences can include a promoter.
  • Homologs Two nucleotide sequences that share a common ancestral sequence and diverged when a species carrying that ancestral sequence split into two species.
  • Translation is the process in which the genetic code carried by messenger RNA (mRNA) directs the synthesis of proteins from amino acids.
  • An agent "inhibits" translation if the amount of protein produced, under a defined set of conditions, is decreased. In one embodiment, translation is inhibited in the presence of an agent if the amount of protein produced is decreased by at least 20%, when compared to the amount of protein produced in the absence of the agent. In one embodiment, translation is inhibited in the presence of an agent if the amount of protein produced is decreased by at least 50%), when compared to the amount of protein produced in the absence of the agent.
  • TS protein is a homodimeric enzyme that has its active site constituted by residues from both subunits of the homodimer.
  • the dimer interface of TS is predominantly formed by interaction between two six-stranded ⁇ -sheet structures. Specifically, in human TS, the interface regions of TS are localized to amino acids 31 -72, 131 - 147, 175-217, and 230-260, and in E.
  • an interface peptide consists of at least five, or at least ten, consecutive amino acids from one of the interface regions of the corresponding (human or E. coli) TS protein.
  • a TS peptide is an interface peptide if it includes at least five, or at least ten, or at least fifteen, sixteen or seventeen consecutive amino acids of the interface region of the TS protein from the that species.
  • a "synthetic" surface interface peptide is an interface peptide produced by genetic engineering techniques, by chemical synthesis, or by cleavage of TS protein in vitro.
  • Isolated An "isolated" biological component (such as a nucleic acid, peptide or protein) has been substantially separated, produced apart from, or purified away from other biological components in the cell of the organism in which the component naturally occurs, i.e., other chromosomal and extrachromosomal DNA and RNA, and proteins.
  • Nucleic acids, peptides and proteins which have been “isolated” include nucleic acids and proteins purified by standard purification methods. The term also embraces nucleic acids, peptides and proteins prepared by recombinant expression in a host cell as well as chemically synthesized nucleic acids.
  • a polypeptide is "isolated” if it has been substantially separated from contaminants, e.g., cellular components (nucleic acids, lipids, carbohydrates, and other polypeptides) that naturally accompany it. Such a polypeptide can also be referred to as “pure” or “homogeneous” or “substantially” pure or homogeneous.
  • a TS peptide is isolated when at least 60-90%) by weight of a sample is composed of the polypeptide, or 95%> or more, or more than 99% of a sample is composed of the peptide.
  • Protein purity or homogeneity is indicated, for example, by polyacrylamide gel electrophoresis of a protein sample, followed by visualization of a single polypeptide band upon staining the polyacrylamide gel; high pressure liquid chromatography; sequencing; or other conventional methods.
  • Mammal This term includes both human and non-human mammals. Similarly, the term “subject” includes both human and veterinary subjects.
  • Mimetic A molecule (such as an organic chemical compound) that mimics the activity of a protein, such as a TS peptide that binds TS mRNA, or variants or fusions thereof.
  • Peptidomimetic and organomimetic embodiments are within the scope of this term, whereby the three-dimensional arrangement of the chemical constituents of such peptido- and organomimetics mimic the three-dimensional arrangement of the peptide backbone and component amino acid sidechains in the peptide, resulting in such peptido- and organomimetics of the peptides having substantial specific inhibitory activity or agonist activity.
  • a pharmacophore is an idealized, three-dimensional definition of the structural requirements for biological activity.
  • Peptido- and organomimetics can be designed to fit each pharmacophore with current computer modeling software (using computer assisted drug design or CADD). See Walters, "Computer-Assisted Modeling of Drugs," in Klegerman & Groves, eds., Pharmaceutical Biotechnology, Interpharm Press: Buffalo Grove, IL, pp. 165-174, 1993 and Principles of Pharmacology (ed. Munson), chapter 102, 1995, for a description of techniques used.
  • a mimetic mimics the binding of a TS peptide or protein to TS mRNA.
  • a mimetic mimics an aspect of the bimolecular interaction of a TS peptide and TS mRNA.
  • Neoplasia The process of tumor formation; the proliferation of cells under conditions that would not elicit similar growth in normal (wild type) cells.
  • An "anti- neoplastic agent” is an agent that decreases the proliferation of neoplastic cells or the growth or metastasis of a tumor.
  • a “neoplastic disorder” is a disorder that involves the proliferation of cells under conditions that would not elicit similar growth in normal (e.g. non-transformed) cells.
  • Nucleic acid A deoxyribonucleotide or ribonucleotide polymer in either single or double stranded form, and unless otherwise limited, encompasses known analogues of natural nucleotides that hybridize to nucleic acids in a manner similar to naturally occurring nucleotides.
  • Oligonucleotide A linear polynucleotide sequence of up to about 200 nucleotide bases in length, for example a polynucleotide (such as DNA or RNA) which is at least 6 nucleotides, for example at least 15, 50, 100 or even 200 nucleotides long.
  • Operably linked A first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence.
  • a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence.
  • operably linked DNA sequences are contiguous and, where necessary to join two protein coding regions, in the same reading frame.
  • Peptide A chain of amino acids of between 3 and 30 amino acids in length. In one embodiment, a peptide is from about 10 to about 25 amino acids in length. In yet another embodiment, a peptide is from about 16 to about 23 amino acids in length. In yet another embodiment, a peptide is about 17 amino acids in length.
  • the present invention includes biologically active peptides that bind TS mRNA.
  • the peptides of the invention include synthetic embodiments of peptides described herein.
  • analogues non-peptide organic molecules
  • derivatives chemically functionalized peptide molecules obtained starting with the disclosed peptide sequences
  • variants homologs of these proteins that specifically bind TS mRNA
  • Each peptide of the invention is comprised of a sequence of amino acids, which may be either L- and/or D- amino acids, naturally occurring and otherwise.
  • Peptides may be modified by a variety of chemical techniques to produce derivatives having essentially the same activity as the unmodified peptides, and optionally having other desirable properties.
  • carboxylic acid groups of the protein may be provided in the form of a salt of a pharmaceutical ly-acceptable cation or esterified to form a C,-C 16 ester, or converted to an amide of formula NR,R 2 wherein R, and R 2 are each independently H or C
  • Amino groups of the peptide may be in the form of a pharmaceutically-acceptable acid addition salt, such as the HC1, HBr, acetic, benzoic, toluene sulfonic, maleic, tartaric and other organic salts, or may be modified to C,-C 16 alkyl or dialkyl amino or further converted to an amide.
  • a pharmaceutically-acceptable acid addition salt such as the HC1, HBr, acetic, benzoic, toluene sulfonic, maleic, tartaric and other organic salts
  • Hydroxyl groups of the peptide side chains may be converted to C,-C l6 alkoxy or to a C,-C l6 ester using well-recognized techniques.
  • Phenyl and phenolic rings of the peptide side chains may be substituted with one or more halogen atoms, such as fluorine, chlorine, bromine or iodine, or with C,-C l6 alkyl, C,-C 16 alkoxy, carboxylic acids and esters thereof, or amides of such carboxylic acids.
  • Methylene groups of the peptide side chains can be extended to homologous C 2 -C 4 alkylenes.
  • Thiols can be protected with any one of a number of well-recognized protecting groups, such as acetamide groups.
  • protecting groups such as acetamide groups.
  • Peptidomimetic and organomimetic embodiments are also within the scope of the present invention, whereby the three-dimensional arrangement of the chemical constituents of such peptido- and organomimetics mimic the three-dimensional arrangement of the peptide backbone and component amino acid side chains, resulting in such peptido- and organomimetics of the proteins of this invention having measurable or enhanced ability to bind TS mRNA.
  • a pharmacophore is an idealized, three-dimensional definition of the structural requirements for biological activity.
  • Peptido- and organomimetics can be designed to fit each pharmacophore with current computer modeling software (using computer assisted drug design or CADD).
  • a mimetic mimics the binding of a TS peptide or TS protein to TS mRNA.
  • a mimetic mimics the bimolecular complex of a TS peptide or protein and TS mRNA.
  • Pharmaceutical agent or drug a chemical compound or composition capable of inducing a desired therapeutic or prophylactic effect when properly administered to a subject.
  • compositions and formulations suitable for pharmaceutical delivery of the TS peptides herein disclosed are conventional. Remington 's Pharmaceutical Sciences, by E. W. Martin, Mack Publishing Co., Easton, PA, 15th Edition (1975), describes compositions and formulations suitable for pharmaceutical delivery of the TS peptides herein disclosed.
  • parenteral formulations usually comprise injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle.
  • pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle.
  • physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like
  • solid compositions e.g. , powder, pill, tablet, or capsule forms
  • conventional non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate.
  • compositions to be administered can contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate.
  • non-toxic auxiliary substances such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate.
  • Polynucleotide A linear nucleic acid sequence of any length. Therefore, a polynucleotide includes molecules which are no more than 15, 50, 100, 200 nucleotides in length (oligonucleotides) and also nucleotides as long as a full length cDNA.
  • a "TS polynucleotide” encodes a TS peptide.
  • a promoter is an array of nucleic acid control sequences that directs transcription of a nucleic acid.
  • a promoter includes necessary nucleic acid sequences near the start site of transcription, such as, in the case of a polymerase II type promoter, a TATA element.
  • a promoter also optionally includes distal enhancer or repressor elements which can be located as much as several thousand base pairs from the start site of transcription. Both constitutive and inducible promoters, are included (see e.g., Bitter et al., Methods in Enzymology 153:516-544, 1987).
  • promoters include promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or from mammalian viruses (e.g. , the retrovirus long terminal repeat; the adenovirus late promoter; the vaccinia virus 7.5K promoter) can be used. Promoters produced by recombinant DNA or synthetic techniques can also be used. A polynucleotide encoding a TS peptide that binds TS mRNA can be inserted into an expression vector that contains a promoter sequence which facilitates the efficient transcription of the inserted genetic sequence of the host.
  • the expression vector typically contains an origin of replication, a promoter, as well as specific nucleic acid sequences that allow phenotypic selection of the transformed cells
  • purified does not require absolute purity; rather, it is intended as a relative term.
  • a purified peptide preparation is one in which the peptide or protein is more enriched than the peptide or protein is in its natural environment within a cell.
  • a preparation is purified such that the protein or peptide represents at least 50%) of the total peptide or protein content of the preparation.
  • a recombinant nucleic acid is one that has a sequence that is not naturally occurring or has a sequence that is made by an artificial combination of two otherwise separated segments of sequence. This artificial combination is often accomplished by chemical synthesis or, more commonly, by the artificial manipulation of isolated segments of nucleic acids, e.g., by genetic engineering techniques.
  • a recombinant protein is one encoded for by a recombinant nucleic acid molecule.
  • Specific binding agent An agent that binds substantially only to a defined target.
  • a TS peptide is a specific binding agent that binds substantially only to
  • Sequence identity The similarity between amino acid sequences is expressed in terms of the similarity between the sequences, otherwise referred to as sequence identity. Sequence identity is frequently measured in terms of percentage identity (or similarity or homology); the higher the percentage, the more similar the two sequences are. Homologs or variants of TS or a specific TS peptide that binds TS mRNA, disclosed herein, will possess a relatively high degree of sequence identity when aligned using standard methods.
  • NCBI Basic Local Alignment Search Tool (BLASTTM) (Altschul et al., J. Mol. Biol. 215:403-410, 1990) is available from several sources, including the National Center for Biotechnology Information (NCBI, Bethesda, MD) and on the Internet, for use in connection with the sequence analysis programs blastp, blastn, blastx, tblastn and tblastx.
  • Variants of TS, or variants of a TS peptide that bind TS mRNA are typically characterized by possession of at least 50%> sequence identity counted over the full length alignment with the amino acid sequence of TS or a TS peptide that binds TS mRNA using the NCBI Blast 2.0, gapped blastp set to default parameters.
  • the Blast 2 sequences function is employed using the default BLOSUM62 matrix set to default parameters, (gap existence cost of 11 , and a per residue gap cost of 1).
  • the alignment is performed using the Blast 2 sequences function, employing the PAM30 matrix set to default parameters (open gap 9, extension gap 1 penalties). Proteins with even greater similarity to the reference sequences will show increasing percentage identities when assessed by this method, such as at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 90%, or at least 95%), or 98%> sequence identity.
  • homologs and variants When less than the entire sequence is being compared for sequence identity, homologs and variants will typically possess at least 75% sequence identity over short windows of 10-20 amino acids, and may possess sequence identities of at least 85%> or at least 90%>, 95%, or 98% depending on their similarity to the reference sequence.
  • Therapeutically active molecule A molecule that binds TS mRNA, and inhibits translation of the mRNA, as measured by clinical response (for example increase in the efficacy of a chemotherapeutic agent, or measurable reduction of tumor burden).
  • Therapeutically active molecules can also be made from nucleic acids. Examples of nucleic acid based therapeutically active molecules are a nucleic acid sequence that encodes a TS peptide that binds TS mRNA, wherein the nucleic acid sequence is operably linked to a control element such as a promoter.
  • Therapeutically effective dose A dose sufficient to prevent advancement, or to cause regression of the disease, or which is capable of relieving symptoms caused by the disease, such as fever, pain, decreased appetite or cachexia associated with malignancy, or is sufficient to increase the efficacy of another agent, such as a chemotherapeutic agent.
  • Thymidylate synthase An enzyme that catalyzes the final step in the de novo synthesis of deoxy-thymidine monophosphate (dTMP) using the substrate, dUMP, and a cofactor, 5,10-methylene tetrahydrofolate. Because of its essential role in DNA replication, human TS is an anticancer drug target and TS from infectious pathogens are infectious disease drug targets.
  • the sequence of thymidylate synthase protein is known for many species, including human, rat, and E. coli, amongst others. In addition the sequence of nucleic acids encoding TS is also known for many species, including human (e.g. see Genbank Accession Nos.
  • NM 001071, BF109634, X02308 feline (Genbank Accession No. AH 009262), leishmainia (Genbank Accession No. AC078892), rat (Genbank Accession No. L12138), yeast (Genbank Accession No. AL 109770), mouse (Genbank Accession No. M29309) and ⁇ .coli (Genbank Accession No. J01710; all references for TS sequences incorporated herein by reference).
  • Thymidylate synthase peptide A peptide (for example, a peptide consisting essentially of from about 10 to about 25 consecutive amino acids) of a thymidylate synthase protein.
  • a "human thymidylate synthase peptide” is at least 10 consecutive amino acids of a human thymidylate synthase protein encoded by a nucleic acid isolated or derived from a human cell.
  • an "E. coli thymidylate synthase peptide” is at least 10 consecutive amino acids of a thymidylate synthase protein encoded by a nucleic acid isolated or derived from E. coli.
  • a TS peptide is from about 10 to about 25 consecutive amino acids of a TS protein. In yet another embodiment, a peptide is from about 16 to about 23 consecutive amino acids of a TS protein. In yet another embodiment, a peptide is about 17 consecutive amino acids of a TS protein.
  • a TS peptide can be naturally occurring, produced using genetic engineering (e.g. cloning) methodology, or synthesized.
  • Transduced and Transformed A virus or vector "transduces" of
  • transformation encompasses all techniques by which a nucleic acid molecule might be introduced into such a cell, including transfection with viral vectors, transformation with plasmid vectors, and introduction of naked DNA by electroporation, lipofection, and particle gun acceleration.
  • transfected cell is a cell into which has been introduced a nucleic acid molecule by molecular biology techniques.
  • transfection encompasses all techniques by which a nucleic acid molecule might be introduced into such a cell, including transfection with viral vectors, transformation with plasmid vectors, and introduction of naked DNA by electroporation, lipofection, and particle gun acceleration.
  • Transgene An exogenous nucleic acid sequence supplied by a vector.
  • a transgene encodes a TS peptide that binds TS mRNA.
  • Tumor A neoplasm that may be either malignant or non-malignant.
  • Tumors of the same tissue type refers to primary tumors originating in a particular organ (such as breast, prostate, bladder or lung). Tumors of the same tissue type may be divided into tumors of different sub-types (a classic example being bronchogenic carcinomas (lung tumors) which can be an adenocarcinoma, small cell, squamous cell, or large cell tumor).
  • a vector may include nucleic acid sequences that permit it to replicate in the host cell, such as an origin of replication.
  • a vector may also include one or more therapeutic genes and/or selectable marker genes and other genetic elements known in the art.
  • a vector can transduce, transform or infect a cell, thereby causing the cell to express nucleic acids and/or proteins other than those native to the cell.
  • a vector optionally includes materials to aid in achieving entry of the nucleic acid into the cell, such as a viral particle, liposome, protein coating or the like.
  • a vector is a viral vector.
  • Viral vectors include, but are not limited to, retroviral and adenoviral vectors.
  • TS Protein and Binding to TS mRNA TS protein has been shown to regulate its own translational efficiency through an interaction with its mRNA (Chu et al., Proc. Natl. Acad. Sci. U.S.A. 90, 517-521, 1993; Chu et al., Proc Natl Acad Sci USA 88, 8977-8981, 1991 ; Chu et al., J. Biol. Chem. 269, 20289-220293, 1994).
  • the interaction of full-length TS protein with its own mRNA has been demonstrated in both cell-free and intact cell models (Chu et al., Proc. Natl. Acad. Sci. U.S.A.
  • the TS protein of Escherichia coli (E. coli) binds to its own mRNA thus suggesting evolutionary conservation of this autoregulatory phenomenon (Voeller et al., Nucleic Acids Res. 23, 869-875, 1995).
  • the E. coli TS protein also binds to the human TS mRNA.
  • Other levels of control of intracellular TS exist and include regulation at the transcriptional level and possible stabilization of TS protein through interaction of the enzyme with its substrates or inhibitors (Yee et al., Exp. Cell Res. 258, 53-64, 2000; Horie et al., Nucleic Acids Symp. Ser. 34, 77-78, 1995; Kitchens et al., J.
  • the autoregulation of TS translational efficiency occurs through the interaction of TS protein with either or both of two regions on TS mRNA.
  • the first of these two regions includes the translational start sequence and occurs at nucleotides 81-110 and the second lies between nucleotides 450 and 520 in the protein coding region (Chu et al., Proc Natl Acad Sci USA 88, 8977-8981, 1991; Lin et al., Nucleic Acids. Res. 28, 1381-1389, 2000).
  • the first binding region has been predicted to form a stem-loop structure containing at least one bulge-C in the stem (Chu et al., Proc. Natl.
  • TS protein has also been shown to interact with several other mRNAs including c-myc and p53 thus suggesting that TS protein may have broad regulatory functions in addition to its well described catalytic function (Chu et al., Adv. Enzyme Regul. 36, 143-163, 1996; Chu et al., Mol. Cell. Biol. 15, 179-185, 1995; Chu et al., Mol, Cell Biol. 19, 1582- 1594, 1995).
  • peptides As disclosed herein, several peptides have been identified that bind TS mRNA. In one embodiment, the binding of one of these peptides inhibits the translation of TS mRNA.
  • a peptide that binds TS mRNA is a peptide consisting essentially of at least 10 consecutive amino acids of a member of the group consisting of SEQ ID NO: 4 (E. coli 8), SEQ ID NO: 8 (E. coli 25), SEQ ID NO: 10 (E. coli 28) and S ⁇ Q ID NO: 12 (E. coli 33).
  • the peptide is S ⁇ Q ID NO: 4 (E coli 8), S ⁇ Q ID NO: 8 (E. coli 25), S ⁇ Q ID NO: 10 (E. coli 28) and S ⁇ Q ID NO: 12 (E. coli 33).
  • Peptides consisting essentially of from about 10 consecutive amino acids to about 25 amino acids of the peptide interface region of thymidylate synthase protein, are examples of peptides disclosed herein that can be used to inhibit the translation of TS mRNA. These peptides bind to the mRNA encoding thymidylate synthase. In one embodiment, the binding of the peptides inhibits the translation of the mRNA encoding thymidylate synthase.
  • the TS mRNA can be TS mRNA derived from any species, including, but not limited to human (e.g. see Genbank Accession Nos. NM 001071, BF 109634, X02308), feline (Genbank Accession No. AH 009262), leishmania (Genbank
  • the peptides are sequences from the peptide interface region of TS protein.
  • the TS protein can be derived from any species, including, but not limited to, human (SEQ ID NO: 14, see Fig. 2), feline, leishmainia, rat, yeast, and E. coli.
  • the TS peptide that binds TS mRNA is a fragment of at least 10 consecutive amino acids of the peptide interface region of a TS protein or a variant or homolog thereof.
  • a heterologous TS peptide can be used to inhibit translation from a TS mRNA.
  • an E. coli TS peptide can be used to inhibit the translation of a human TS mRNA (e.g. SEQ ID NO: 13, see Fig. 2).
  • a murine TS peptide can be used to inhibit translation of human TS mRNA.
  • a human TS peptide can be used to inhibit the translation of feline TS mRNA.
  • the peptide that binds TS mRNA consists essentially of an amino acid sequence of a least 10 consecutive amino acids of a member of the group consisting of SEQ ID NO: 4 (E. coli 8), S ⁇ Q ID NO: 8 (E. coli 25), S ⁇ Q ID NO: 10 (E coli 28), S ⁇ Q ID NO: 12 (E. coli 33), S ⁇ Q ID NO: 1 (human 9), and S ⁇ Q ID NO: 3 (human 14), S ⁇ Q ID NO: 5 (human 29), S ⁇ Q ID NO: 9 (human 38), and S ⁇ Q ID NO: 11 (human 43).
  • the peptide that binds TS mRNA consists essentially of an amino acid sequence of a least 14 consecutive amino acids of a member of the group consisting of S ⁇ Q ID NO: 4 (E. coli 8), S ⁇ Q ID NO: 8 (E. coli 25), S ⁇ Q ID NO: 10 (E. coli 28), S ⁇ Q ID NO: 12 (E. coli 33), S ⁇ Q ID NO: 1 (human 9), and S ⁇ Q ID NO: 3 (human 14), S ⁇ Q ID NO: 5 (human 29), S ⁇ Q ID NO: 9 (human 38), and S ⁇ Q ID NO: 11 (human 43).
  • the peptide that binds TS mRNA is S ⁇ Q ID NO: 4 (E coli 8), S ⁇ Q ID NO: 8 (E. coli 25), S ⁇ Q ID NO: 10 (E. coli 28), S ⁇ Q ID NO: 12 (E. coli 33), S ⁇ Q ID NO: 1 (human 9), and S ⁇ Q ID NO: 3 (human 14), S ⁇ Q ID NO: 5 (human 29), S ⁇ Q ID NO: 9 (human 38), and S ⁇ Q ID NO: 1 1 (human 43).
  • the fluropyrimidines are an important group of anitneoplastic agents used widely in the treatment of tumors (e.g. gastrointestinal tumors), and are frequently used in fluoropyrimidine based combination chemotherapy.
  • a fluoropyrimidine is 5-fluorouracil (5-FU).
  • 5-FU 5-fluorouracil
  • An important mechanism of 5FU cytotoxicity is through the inhibition of TS.
  • Murine and cell culture models and the malignant tissues of patients with breast cancer have demonstrated relatively rapid 3-5 fold increases in thymidylate synthase (TS) enzyme levels within 24 hours following exposure to fluoropyrimidines in the absence of changes in TS mRNA (Keyomarsi et al., J.
  • the effect of a chemotherapeutic agent e.g. 5-FU
  • a chemotherapeutic agent e.g. 5-FU
  • the peptide binds to a mRNA encoding TS, and inhibits the translation of the mRNA encoding TS. This inhibition of the translation of TS enhances the chemotherapeutic effect of the chemotherapeutic agent.
  • the chemotherapeutic agent is a fluoropyrimidine.
  • the fluoropyrimidine can be included as part of a regimen including a combination of chemotherapeutic agents.
  • a combination of chemotherapeutic agents is a regimen including treatment with 5-FU, cisplatin, and VP-16.
  • the chemotherapeutic agent can be used to treat any neoplastic disorder, including, but not limed to a head and neck cancer, breast cancer, prostate cancer, liver cancer, lung cancer, gastric cancer, colon cancer, or esophageal cancer.
  • method of treating a subject with a neoplastic disorder includes administering to the subject a chemotherapeutic agent and a TS peptide consisting essentially of from about 10 consecutive amino acids to about 25 amino acids of the peptide interface region of TS, wherein the peptide binds to the mRNA encoding TS, and inhibits the translation of the mRNA encoding TS, thereby treating the neoplastic disorder.
  • the method includes administering to the subject a chemotherapeutic agent and a therapeutic nucleic acid sequence comprising a promoter operably linked to a nucleic acid sequence encoding a TS peptide.
  • the TS peptide consists essentially of from about 10 consecutive amino acids to about 25 amino acids of the peptide interface region of TS, wherein the TS peptide binds to the mRNA encoding TS.
  • retroviral vectors For administration of nucleic acids molecules, various viral vectors can be utilized. These vectors include adenovirus, herpes virus, vaccinia, or an RNA virus such as a retrovirus. In one embodiment, the retroviral vector is a derivative of a murine or avian retrovirus. Examples of retroviral vectors in which a single foreign gene can be inserted include, but are not limited to: Moloney murine leukemia virus (MoMuLV), Harvey murine sarcoma virus (HaMuSV), murine mammary tumor virus (MuMTV), and Rous Sarcoma Virus (RSV). When the subject is a human, a vector such as the gibbon ape leukemia virus (GaLV) can be utilized.
  • MoMuLV Moloney murine leukemia virus
  • HaMuSV Harvey murine sarcoma virus
  • MuMTV murine mammary tumor virus
  • RSV Rous Sarcoma Virus
  • GaLV
  • retroviral vectors can incorporate multiple genes. All of these vectors can transfer or incorporate a gene for a selectable marker so that transduced cells can be identified and generated. By inserting a nucleic acid sequence encoding a TS peptide into the viral vector, along with another gene which encodes the ligand for a receptor on a specific target cell, for example, the vector is now target specific. Retroviral vectors can be made target specific by attaching, for example, a sugar, a glycolipid, or a protein. Preferred targeting is accomplished by using an antibody to target the retroviral vector.
  • helper cell lines that contain plasmids encoding all of the structural genes of the retrovirus under the control of regulatory sequences within the LTR. These plasmids are missing a nucleotide sequence which enables the packaging mechanism to recognize an RNA transcript for encapsidation.
  • Helper cell lines which have deletions of the packaging signal include, but are not limited to Q2, PA317, and PA 12, for example. These cell lines produce empty virions, since no genome is packaged. If a retroviral vector is introduced into such cells in which the packaging signal is intact, but the structural genes are replaced by other genes of interest, the vector can be packaged and vector virion produced.
  • NIH 3T3 or other tissue culture cells can be directly transfected with plasmids encoding the retroviral structural genes gag, pol and env, by conventional calcium phosphate transfection. These cells are then transfected with the vector plasmid containing the genes of interest. The resulting cells release the retroviral vector into the culture medium.
  • colloidal dispersion systems include macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes.
  • One colloidal system of this invention is a liposome.
  • Liposomes are artificial membrane vesicles that are useful as delivery vehicles in vitro and in vivo. It has been shown that large uni-lamellar vesicles (LUV), which range in size from 0.2-4.0 ⁇ m can encapsulate a substantial percentage of an aqueous buffer containing large macromolecules.
  • LUV large uni-lamellar vesicles
  • RNA, DNA and intact virions can be encapsulated within the aqueous interior and be delivered to cells in a biologically active form (Fraley et al. , 1981, Trends Biochem. Sci. 6:77, 1981).
  • liposomes In addition to mammalian cells, liposomes have been used for delivery of polynucleotides in plant, yeast and bacterial cells.
  • a liposome In order for a liposome to be an efficient gene transfer vehicle, the following characteristics should be present: (1) encapsulation of the genes of interest at high efficiency while not compromising their biological activity; (2) preferential and substantial binding to a target cell in comparison to non-target cells; (3) delivery of the aqueous contents of the vesicle to the target cell cytoplasm at high efficiency; and (4) accurate and effective expression of genetic information (Mannino et al, Biotechniques 6:682, 1988).
  • the composition of the liposome is usually a combination of phospholipids, particularly high-phase-transition-temperature phospholipids, usually in combination with steroids, especially cholesterol. Other phospholipids or other lipids may also be used.
  • the physical characteristics of liposomes depend on pH, ionic strength, and the presence of divalent cations.
  • lipids useful in liposome production include phosphatidyl compounds, such as phosphatidylglycerol, phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, sphingolipids, cerebrosides, and gangliosides.
  • phosphatidyl compounds such as phosphatidylglycerol, phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, sphingolipids, cerebrosides, and gangliosides.
  • diacylphosphatidyl-glycerols where the lipid moiety contains from 14-18 carbon atoms, particularly from 16-18 carbon atoms, and is saturated.
  • Illustrative phospholipids include egg phosphatidylcholine, dipalmitoylphosphatidylcholine and distearoylphosphatidylcholine.
  • the targeting of liposomes can be classified based on anatomical and mechanistic factors. Anatomical classification is based on the level of selectivity, for example, organ-specific, cell-specific, and organelle-specific. Mechanistic targeting can be distinguished based upon whether it is passive or active.
  • Passive targeting utilizes the natural tendency of liposomes to distribute to cells of the reticulo-endothelial system (RES) in organs which contain sinusoidal capillaries.
  • Active targeting involves alteration of the liposome by coupling the liposome to a specific ligand such as a monoclonal antibody, sugar, glycolipid, or protein, or by changing the composition or size of the liposome in order to achieve targeting to organs and cell types other than the naturally occurring sites of localization.
  • the surface of the targeted delivery system may be modified in a variety of ways.
  • lipid groups can be incorporated into the lipid bilayer of the liposome in order to maintain the targeting ligand in stable association with the liposomal bilayer.
  • Various linking groups can be used for joining the lipid chains to the targeting ligand.
  • This invention involves administering to a subject a therapeutically effective dose of a pharmaceutical composition containing the compounds of the present invention and a pharmaceutically acceptable carrier.
  • Administering the pharmaceutical composition of the present invention may be accomplished by any means known to the skilled artisan.
  • subject is meant any mammal, including a human.
  • the pharmaceutical compositions are preferably prepared and administered in dose units.
  • Solid dose units are tablets, capsules and suppositories.
  • different daily doses are necessary. Under certain circumstances, however, higher or lower daily doses may be appropriate.
  • the administration of the daily dose can be carried out both by single administration in the form of an individual dose unit or else several smaller dose units and also by multiple administration of subdivided doses at specific intervals.
  • compositions according to the invention are in general administered topically, intravenously, orally or parenterally or as implants, but even rectal use is possible in principle.
  • suitable solid or liquid pharmaceutical preparation forms are, for example, granules, powders, tablets, coated tablets, (micro)capsules, suppositories, syrups, emulsions, suspensions, creams, aerosols, drops or injectable solution in ampule form and also preparations with protracted release of active compounds, in whose preparation excipients and additives and/or auxiliaries such as disintegrants, binders, coating agents, swelling agents, lubricants, flavorings, sweeteners or solubilizers are customarily used as described above.
  • compositions are suitable for use in a variety of drug delivery systems.
  • the pharmaceutical compositions according to the invention may be administered locally or systemically. Amounts effective for therapeutic use will, of course, depend on the severity of the disease and the weight and general state of the patient. Typically, dosages used in vitro may provide useful guidance in the amounts useful for in situ administration of the pharmaceutical composition, and animal models may be used to determine effective dosages for treatment of particular disorders.
  • TS peptide will vary depending on the nature and severity of the condition to be treated, the age and condition of the patient and other clinical factors. Thus, the final determination of the appropriate treatment regimen will be made by the attending clinician. Typically, the dose range for a TS peptide will be from about 0.1 ⁇ g/kg body weight to about lOOmg/kg body weight.
  • Suitable ranges include doses of from about 1 ⁇ g/kg to lOmg/kg body weight.
  • the dosing schedule may vary from once a week to daily depending on a number of clinical factors, such as the subject's sensitivity to the TS peptide. In the case of a more aggressive disease it may be preferable to administer doses such as those described above by alternate routes including intravenously or intrathecally. Continuous infusion may also be appropriate.
  • purified therapeutically active molecules are generally combined with a pharmaceutically acceptable carrier.
  • Pharmaceutical preparations may contain only one type of therapeutic molecule, or may be composed of a combination of several types of therapeutic molecules.
  • the nature of the carrier will depend on the particular mode of administration being employed.
  • parenteral formulations usually comprise injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle.
  • conventional non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate.
  • compositions to be administered can contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate.
  • non-toxic auxiliary substances such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate.
  • protein-based pharmaceuticals may be only inefficiently delivered through ingestion.
  • pill-based forms of pharmaceutical proteins may be administered subcutaneously, particularly if formulated in a slow-release composition.
  • Slow-release formulations may be produced by combining the target protein with a biocompatible matrix, such as cholesterol.
  • a biocompatible carrier such as cholesterol.
  • compositions of the present invention may be administered by any means that achieve their intended purpose. Amounts and regimens for the administration of the therapeutic molecules can be determined readily by those with ordinary skill in the clinical art of treating diseases associated with tumor growth. For use in treating these conditions, molecules are administered in an amount effective to inhibit metastasis. Typical amounts initially administered would be those amounts adequate to achieve tissue concentrations at the site of action which have been found to achieve the desired effect in vitro.
  • the peptides or proteins may be administered to a host in vivo, for example through systemic administration, such as intravenous or intraperitoneal administration. Also, the peptides or nucleic acids may be administered intralesionally: i.e. the peptide or protein is injected directly into the tumor or affected area.
  • a method of identifying an agent that binds a mRNA encoding TS includes contacting the mRNA encoding TS, or a cell in vitro comprising the mRNA encoding TS, with a TS peptide and the agent.
  • the TS peptide consists essentially of from about 10 consecutive amino acids to about 25 amino acids of the peptide interface region of TS and with the agent.
  • the peptide consists essentially of an amino acid sequence of a least 10 consecutive amino acids of a member of the group consisting of SEQ ID NO: 4 (E. coli 8), SEQ ID NO: 8 (E. coli 25), S ⁇ Q ID NO: 10 (E.
  • S ⁇ Q ID NO: 12 E. coli 33
  • S ⁇ Q ID NO: 1 human 9
  • S ⁇ Q ID NO: 3 human 14
  • S ⁇ Q ID NO: 5 human 29
  • S ⁇ Q ID NO: 9 human 38
  • S ⁇ Q ID NO: 11 human 43
  • the binding of the agent to the mRNA encoding TS encoding TS is then evaluated by any means known to one of skill in the art.
  • the agent is directly labeled, such as by a radioactive, enzymatic, or fluorescent label.
  • assays are performed (e.g. gel shift assays, ⁇ LISA assays) that demonstrate the binding of the agent to the TS mRNA.
  • the binding of the agent to mRNA encoding TS indicates the ability of the agent to inhibit translation of TS mRNA.
  • the assay is a competitive binding assay, wherein the TS peptide is labeled.
  • the binding of a TS peptide to TS mRNA is evaluated in the presence of an agent.
  • the binding of the TS peptide is compared to a control, such as a standard value.
  • the binding of the TS peptide to TS mRNA is evaluated in the presence and the absence of an agent.
  • the amount of binding of the TS peptide to the TS mRNA in the presence of the agent is compared to the amount of binding of the TS peptide to the TS mRNA in the absence of the agent.
  • a decrease in the ability of the TS peptide to bind TS mRNA in the presence of the agent demonstrates that the agent can bind TS mRNA.
  • a decrease in the ability of the TS peptide to bind TS mRNA in the presence of an agent is at least a 20%> decrease, as compared to a control. In another embodiment, a decrease in the ability of the TS peptide to bind TS mRNA in the presence of an agent is at least a 50%> decrease, as compared to a control. In an additional embodiment, a decrease in the ability of the TS peptide to bind TS mRNA in the presence of an agent is at least a 70% decrease, as compared to a control.
  • mRNA encoding TS or a fragment thereof can be immobilized on a substrate (e.g., a column or filter) and a solution containing a TS peptide, such as a synthetic interface peptide can be introduced to the column to allow binding. Then a solution including the test compound(s) is introduced to the column under conditions that are permissive for binding. The substrate is then washed with a solution to remove unbound or weakly bound molecules. A second wash may then elute those compounds that strongly bound to the immobilized mRNA.
  • the test compounds may be immobilized and a solution containing TS mRNA and/or a TS peptide may be contacted with the column, filter or other substrate.
  • Radioactively labeled RNA transcripts were made by inclusion of [ PJCTP (spec, act. 200 Ci/mmol) in the reaction.
  • a 30-nt RNA sequence corresponding to nt 81- 1 10 that includes the translational start site was also used as an RNA probe.
  • the smaller RNA probe was prepared by end-labeling the RNA with [ 32 P]ATP (3000 Ci/mmol) using T4 polynucleotide kinase (Promega).
  • a 150 bp fragment of Actin RNA was synthesized with T7 RNA polymerase following linearization of the PGEM4Z plasmid with AVAL In vitro transcription reactions were performed as described above.
  • RNA-Protein Binding Assay - RNA protein binding experiments were performed using an electrophoretic gel mobility shift assay (EMSA) (Chu et al.,
  • Radiolabeled TS RNA (1-2.2 fmol, 100,000 cpm) was incubated with TS peptides (1-2 ⁇ g) in a reaction mixture containing lOmM Hepes pH 7.4, 40 mM KC1, 3mM MgCl 2 , 250 mM 2-mercaptoethanol, 0.1 unit/ ⁇ l Rnase Inhibitor (Eppendorf), and 5%> (v/v) glycerol for 15 min at room temperature.
  • TS full-length RNA Rnase TI (9 units; Eppendorf, Long Island, NY) was added for 10 min, followed by heparin sulfate (5 mg/ml, (Sigma, St. Louis, MO) for an additional 10 min.
  • the reaction samples were resolved on a 4%> nondenaturing polyacrylamide gel (acrylamide/methylenebisacrylamide ratio, 60:1), dried, and visualized by autoradiography.
  • the EMSA was also performed with the TS 30-mer RNA with the same method as the full-length probe but without RnaseTI.
  • SPA Assay - A modified version of the Scintillation Proximity Assay (SPA) developed by Amersham (Piscataway, NJ) to measure the activity of the MAP kinase enzyme was used to complement the electrophoretic gel mobility shift assay (McDonald et al., Anal. Biochem. 268, 318-329, 1999).
  • TS peptide (lug) was incubated with TS 30-mer RNA probe (100,000 cpm) in the same reaction mix as used in the gel shift assay for 15 min at RT.
  • TS peptide Synthesis Two different sets of TS peptides were synthesized by Chiron Mimotopes (Raleigh, NC).
  • the first set for the Human TS protein consisted of 61 17-mer peptides which spanned the entire length of the protein with an overlap of 12 residues per peptide.
  • the peptides were biotinylated at the amino terminus through a serine-glycine-serine-glycine spacer.
  • the second set synthesized in the same manner as the human consisted of 49 17-mer peptides which spanned the entire E. coli TS protein.
  • Goat anti- mouse immunoglobin was purchased from BIO-RAD (Hercules, CA). TCA was purchased from Mallinkrodt Inc. (Paris, KY). Biosafe II scintillation cocktail was purchased from RPI Inc. (Mount Prospect, IL). Protein A Agarose beads were purchased from Invitrogen Corp (Carlsbad, CA).
  • Example 2 Human TS peptides that bind human TS mRNA Using the RNA EMSA, it was first determined which of the 61 human peptides (see Example 1 , above) were able to bind the radiolabeled full-length human TS mRNA. Five of the 61 human peptides were found to be positive for binding to the mRNA including peptides 9, 14, 29, 38, and 43. The sequence and location of these peptides within the full-length protein are illustrated in Fig. 1 A and highlighted on the ribbon structure of one of the monomeric units of the E. coli TS homodimeric protein shown in Fig. IB. The human TS peptides are superimposed onto the E. coli TS structure, which has been shown to be very similar to the human TS structure (Schiffer et al., Biochemistry 34, 16279-87, 1995).
  • a gel shift assay was used to confirm the binding ability of these five peptides to the human TS mRNA, as well as to demonstrate which peptides are non- binding peptide.
  • a control reaction which contains TS mRNA but not peptide, was also used as a control, and the relative complex quantitation was determined by densitometry.
  • a binding titration of peptides 9 and 14 using 0.5, 1, and 2.5 ⁇ g of peptide was performed. In these titration experiments, the signal intensity increased in proportion to the amount of the peptide used in the reaction.
  • the positive binding peptides were also tested for their ability to bind the shorter TS 30-mer RNA probe (nt 81-110) in the EMSA. All of the peptides that bound the full-length protein also bound to the TS 30-mer RNA.
  • E. coli TS protein was shown to bind to human TS mRNA,.
  • a second set of peptides derived from the E. coli TS protein were synthesized and tested for their ability to bind the human TS RNA.
  • any E. coli peptides that bound to the human mRNA and that matched conserved binding regions on the human protein might represent important TS protein/RNA contacts.
  • four E. coli peptides were found to bind the human TS mRNA and included peptides 8, 25, 28, and 33.
  • binding peptides Of these four binding peptides, three of them each corresponded to one of the five human TS peptides that bound to the human TS mRNA.
  • the two E. coli peptides whose sequences corresponded to the human binding peptides 9 and 29, did not bind theTS mRNA.
  • the amino acid sequences of the binding human and E. coli peptides are illustrated in Table 1 for comparison. Table I. conserveed regions of the human and E. coli TS peptides that bind human TS mRNA ⁇
  • E.coli2 ⁇ b GPVYGKQWRAQP TPTDGR (SEQIDNO:6) Human35* L Q R V I D TI K T N P D D RRI (SEQIDNOJ)
  • a Human and E. coli peptides are listed in pairs that represent identical regions of the full-length protein; where either both or a least one of the peptides bind the TS mRNA as determined by a electrophoretic mobility shift assay.
  • the charge of the peptide may play a non-specific role in the binding of peptides to the TS RNA
  • the data show that the binding peptides had various Pi values (isoelectric focusing point) ranging from 6.1 to 11.2 suggesting that non-specific charge interactions are not responsible for the protein-RNA binding.
  • Example 4 Specificity of the Interaction of TS Peptides with TS mRNA
  • the scintillation proximity assay was used to test the binding of the positive peptides to TS RNA.
  • SPA scintillation proximity assay
  • a smaller 30-mer TS RNA probe was used in place of the full-length TS RNA used in the EMSA.
  • Table II peptides that were able to bind RNA in the gel shift were also found to bind to the human TS mRNA in the SPA assay.
  • Table II Human and E. coli peptide binding in electric mobility shift and scintillation proximity assays and their ability to compete for binding with TS and ⁇ -Actin RNA.
  • Each of the six regions defined by either the binding human and/or E. coli peptides lie at the interface of the two monomeric subunits of the dimeric TS protein.
  • GenBank data base was scanned for sequence similarities, and it was determined that these particular regions are very highly conserved amongst 11 different species whose TS protein has been sequenced (Benson et al., Nucl. Acids Res. 28, 15-18, 2000). These species include Human, E. coli, Mouse, Human herpesvirus 8, Bacteriophage T4, Lactobacillus casei, Saccharomyces cerevisiae, Candida albicans, Cryptococcus neoformans, Plasmodium berghei, Leishmania major, and Soy plant.
  • each of the human binding peptide regions except human peptide 9 which was the weakest binder and whose E. coli counterpart did not bind, contain at least one arginine that is absolutely conserved amongst the TS of all 11 species.
  • the conserved arginine in the different peptide regions is underlined in the sequence illustrated in Figure IB.
  • Other human peptides including 25 and 34 that contain arginine were not able to bind TS mRNA, sugggesting that binding is not the result of simply the presence of arginine, but depends as well on its context.
  • Five of the identified peptides contain at least one arginine that is evolutionarily highly conserved across the TS of at least 1 1 different species from bacteriophage too human. These conserved aginines are all situated on the interface region of the protein. Furthermore, equilibrium dialysis experiments demonstrate the coexistence of monomeric and dimeric forms of the TS protein in solution. In addition, three of these highly conserved peptides of 61 human and 49 E. coli peptides tested, overlapped in their ability to specifically bind TS mRNA. These data, taken together support the importance of these peptide areas as potential mRNA contact points.
  • Peptides derived from the 30-mer mRNA binding region are useful as clinical reagents designed to limit the intracellular level of TS protein, which is associated with the clinical use of TS inhibitors, thus sensitizing malignant cells to the effects of these cytotoxins.
  • this molecular model can be used to explore chemical libraries to identify specific clinically useful small molecule inhibitors of TS mRNA that can be used as malignant cell sensitizers in conjunction with commercially available TS inhibitors.
  • TS protein was prepared from a plasmid encoding human TS. A bacterial culture containing the TS plasmid was grown. After pelleting the disrupted bacteria, the protein was purified using a 30- 65%) (NH 4 ) 2 SO 4 fraction and the final pellet was frozen for further purification.
  • the TS protein was further purified and isolated by suspending the pellet in 50mM Tris, pH 7.5, 1 mM EDTA, and 0.2%> 2-mercaptoethanol (Buffer A).
  • Buffer A The protein solution was loaded onto a Blue Sepharose column equilibrated with Buffer A and the column was washed with the same buffer, followed by elution of the protein with Buffer B (50 mM Tris, 1M KCL, 1 MM EDTA, 0.2% 2-me, ph 7.5). Fractions were assayed for TS activity and pooled.
  • the pooled mix was dialyzed overnight at 4° against Buffer C (50 mM Tris, pH .0, 1 mM EDTA, 12 mM 2-me, 0.1% TritonX and 10%) glycerol). The sample was then loaded onto a Q-sepharose fast flow column equilibrated with Buffer C. Three volumes of 0-50 mM KC1 gradient, followed by 10 volumes of a 50-150 mM KC1 gradient in Buffer C was passed through the column as fractions were collected. The fractions were assayed for TS activity and the peak fractions were pooled, and concentrated. The purified protein product (TS B) had a concentration of 0.97 mg/ml.
  • Buffer C 50 mM Tris, pH .0, 1 mM EDTA, 12 mM 2-me, 0.1% TritonX and 10%
  • a lower MW form of TS could pass through a membrane with a specific exclusion size of 50 kD. Since the MW of a monomeric form of TS is approximately 35,000, a 50,000 MW cut-off cellulose ester membrane would be expected to exclude the dimeric form of the enzyme while allowing passage of the monomeric form.
  • BSA and Carbonic Anhydrase were tested in the dialysis chamber to determine the sensitivity and accuracy of the 50,000 MW cut-off membrane. After 72 hours of dialysis at 4°C, the BSA with a MW of 66,000 did not pass through the membrane, but the Carbonic Anhydrase with a MW of 32,000 equilibrated on both sides of the membrane.
  • Western blot analysis all protein samples were resolved on a 12.5% SDS polyacrylamide gel and electroblotted onto a nitrocellulose membrane as previously described (Chu et al, Proc Natl Acad Sci. U.S.A., 88, 8977-8981, 1991).
  • the membrane was incubated with Blotto blocking solution (5%> nonfat milk, 10 mM Tris, 0.01%) thimerosol) for 1 hr and then incubated with the TS monoclonal antibody, TS 106, (1-200 dilution in Blotto) for 1 hr at room temperature.
  • Blotto blocking solution 5%> nonfat milk, 10 mM Tris, 0.01%
  • TS 106 (1-200 dilution in Blotto
  • the blots were washed with IX PBS/0.02%> Tween and incubated with goat anti-mouse immunoglobin (1-600 fold dilution) for 1 hr at room temperature. After the washing was complete, the blots were subjected to a chemiluminescent substrate (Pierce Chemicals, Rockford, IL) for protein signal detection.
  • the Western blot analysis revealed a band at 35 kD, indicating the presence of monomeric TS. As expected with a denaturing gel, a band was also present in the lane with the sample from cell A that contained the original TS sample. This dialysis experiment was repeated on three separate occasions. For all three experiments (with a dialysis time ranging from 72 hr to 115 hr), the TS B protein was able to pass through the 50,000 MW cut-off membrane. The average percent of the starting material that was able to pass through the membrane was 40%>.
  • TS protein was incubated with a TS polyclonal antibody, TS547, at a 1- 10 dilution in 0.1 M Tris pH7.0, 0.15 M NaCl, 5 mM EDTA for 1 hour at 4° C on a rotating shaker.
  • Protein A agarose beads (75 ⁇ l, Invitrogen Corp., Carlsbad, C A) were then added to the protein antibody mix and incubated for an additional hour at 4° C with rotating. The sample was centrifuged and the resulting pellet washed four times with buffer (120 mM NaCl). The TS protein was then eluted with a mass spectrometric solution (alpha-cyano-4-hdyroxy-cinnamic acid) and analyzed by Matrix Assisted Laser Desorption/Ionization (MALDI) mass spectrometry on a Voyager DE-Pro mass spectrometer (Perspective Biosystems, Framingham, MA). The peaks detected on the spectra indicate the MW of the various proteins present in the immunoprecipitated sample.
  • MALDI Matrix Assisted Laser Desorption/Ionization
  • a TS protein sample was incubated with the TS 547 antibody.
  • a TS protein sample was incubated with an anti-tubulin antibody or buffer (no antibody) as controls. Only the spectra from the sample containing TS protein and TS 547 antibody demonstrated a significant peak representing a protein with a MW of 36,911 daltons. This data lent additional support for the existence of a monomeric form of TS.
  • Each of the six regions defined by either the binding human and/or E. coli peptides lie at the interface of the two monomeric subunits of the dimeric TS protein. Given that the interface region would be expected to have limited access to macromolecules such as mRNA, it was critical to demonstrate the presence of monomeric TS in solution. As disclosed herein, two separate experimental systems were employed to demonstrate that a monomeric form of TS protein exists in solution, even though the dimeric form is required for enzymatic function. The equilibrium dialysis experiment shows specific passage of a 35,000 dalton polypeptide that retains full catalytic activity and thus does not represent a degraded portion of dimeric TS enzyme.
  • the inability of the 66 kD albumin protein to pass through the membrane supports the ability of the membrane to exclude dimeric TS.
  • the identification of a 36,911 MW protein polypeptide precipitated by anti-TS antibody by mass spectroscopy further supports the presence of the monomeric form of TS.
  • Translation reactions (final volume, 30 ⁇ l) are performed using a rabbit reticulocyte protein-processing in vitro translation system (New England Nuclear, Boston, MA) as previously described (Chu et al., PNAS USA, 88:8977-8981, 1991). Translation reactions are also performed using either the wheat germ extract system (Promega, Madison, WI) or the E.coliS30 prokaryotic in vitro translation system (Promega, Madison, WI) according to the Promega protocol.
  • Translation protein products are analyzed by SDS-PAGE (12.5% acrylamide) according to the method of Laemmli (Laemmli, Nature, 227:680-685, 1972), and the gels are processed as previously described (Chu et al., PNAS USA, 88:8977-8981, 1991). After drying for two hours, the translation products are visualized by autoradiography.
  • TS peptide The effect of a TS peptide on the translation of TS mRNA is next determined. With the addition of decreasing concentrations of TS, there is a dose-dependent inhibition of TS mRNA translation. This inhibitory effect of TS peptide appears to be specific in that the translation of unrelated mRNAs including human chromogranin and human folate receptor are unaffected. Similar studies are performed using the wheat germ in vitro translation system which confirms that the inhibition of TS mRNA translation by TS peptides is specific. An S30 prokaryotic in vitro coupled transcription-translation system is also employed to test more directly the specificity of TS regulation. When linearized full-length TS cDNA was included in the S30 extract, the corresponding TS protein is synthesized.
  • RNA electrophoretic gel mobility shift assays are also performed using previously published methods (Chu et al., PNAS USA, 88:8977-8981, 1991 ; Chu et al., PNAS USA, 90:517-521, 1993; Leibold et al., PNAS USA, 85:2171-2175, 1988) to identify a TS binding peptide or mimetic.
  • radiolabeled RNA (1- 2.2 fmol; 100 000 c.p.m.) is incubated with recombinant TS peptide or the mimetic (3 pmol) in a reaction mixture containing 10 mM HEPES, pH 7.4, 3 mM MgCl 2 , 40 mM KC1, 5% (vol/vol) glycerol and 250 mM 2-mercaptoethanol (total volume,
  • Competition experiments are performed by incubating TS peptide or a mimetic (3 pmol) and 32 P-radiolabeled TS mRNA (2.2 fmol; 100 000 c.p.m.) in the presence of various unlabeled competitor RNAs, at concentrations in 0-1000-fold molar excess over that of radiolabeled TS mRNA.
  • the relative binding affinity (IC 50 ) of each competitor RNA is measured in terms of the concentration at which specific binding of radiolabeled full-length TS mRNA to TS peptide or mimetic is inhibited by 50%.
  • Each competition experiment is performed three to five times. Quantitation is performed using densitometry on a ScanJet Plus scanner (Hewlett- Packard) using NIH IMAGE 1.36 software (Wayne Rasband, National Institutes of Mental Health, Bethesda, MD).
  • the EMSA assay is performed in the presence of a TS peptide and competitor. The disappearance of a protected RNA band is assessed.

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Abstract

L'invention porte sur un procédé d'inhibition de la traduction de l'ARNm codant pour la thymidylate synthase (TS), consistant à mettre en contact l'ARN m ou une cellule l'exprimant avec un peptide consistant essentiellement en environ 10 à 25 acides aminés de la région interface du peptide de la TS, ledit peptide se fixant audit ARNm et en inhibant la traduction. L'invention porte également sur des procédés d'identification d'agents se fixant à l'ARNm de la TS et en inhibant la traduction, et sur une méthode de traitement d'un sujet à l'aide d'un peptide consistant essentiellement en environ 10 à 25 acides aminés de la région interface du peptide de la TS.
PCT/US2002/006634 2001-03-07 2002-03-05 Peptides de thymidylate synthase se fixant a l'arnm de la thymidylate synthase WO2002072753A2 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
ITMI20092117A1 (it) * 2009-12-01 2011-06-02 Univ Degli Studi Modena E Reggio Emilia Struttura del cristallo del complesso di timidilato sintetasi (ts) con un ligando
WO2011067624A1 (fr) 2009-12-01 2011-06-09 Universita' Degli Studi Di Modena E Reggio Emilia Peptides se liant à l'interface de dimère de la thymidylate synthase pour le traitement du cancer

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
CHU, E. ET AL.: 'Autoregulation of human thymidylate synthase messenger RNA translation by thymidylate synthase' PROC. NATL. ACAD SCI. USA vol. 8, October 1991, pages 8977 - 8981, XP002081371 *
CHU, E. ET AL.: 'Identification of an RNA binding site for human thymidylate synthase' PROC. NATL. ACAD. SCI. USA vol. 90, January 1993, pages 517 - 521, XP002081370 *
LIN, X. ET AL.: 'Characterization of a cis-acting regulatory element in the protein coding region of thymidylate synthase mRNA' NUCLEIC ACIDS RESEARCH vol. 28, no. 6, 15 March 2000, pages 1381 - 1389, XP002984376 *
VOELLER, D.M. ET AL.: 'The identification of thymidylate synthase peptide domains located in the interface region that bind thymidylate synthase mRNA' BIOCHEM. BIOPHYS. RES. COMUN. vol. 297, July 2002, pages 24 - 31, XP002984377 *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
ITMI20092117A1 (it) * 2009-12-01 2011-06-02 Univ Degli Studi Modena E Reggio Emilia Struttura del cristallo del complesso di timidilato sintetasi (ts) con un ligando
WO2011067715A1 (fr) 2009-12-01 2011-06-09 Universita' Degli Studi Di Siena Structure cristalline d'un complexe de thymidylate synthase (ts) avec un ligand
WO2011067624A1 (fr) 2009-12-01 2011-06-09 Universita' Degli Studi Di Modena E Reggio Emilia Peptides se liant à l'interface de dimère de la thymidylate synthase pour le traitement du cancer
US20120302511A1 (en) * 2009-12-01 2012-11-29 Maria Paola Costi Peptides binding to the dimer interface of thymidylate synthase for the treatment of cancer
US8916679B2 (en) * 2009-12-01 2014-12-23 Universita′ degli Studi di Modena e Reggio Emilia Peptides binding to the dimer interface of thymidylate synthase for the treatment of cancer

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