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WO1995003833A1 - Oligomeres modulant la proteine kinase c - Google Patents

Oligomeres modulant la proteine kinase c Download PDF

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Publication number
WO1995003833A1
WO1995003833A1 PCT/US1994/008465 US9408465W WO9503833A1 WO 1995003833 A1 WO1995003833 A1 WO 1995003833A1 US 9408465 W US9408465 W US 9408465W WO 9503833 A1 WO9503833 A1 WO 9503833A1
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PCT/US1994/008465
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Nicholas M. Dean
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Isis Pharmaceuticals, Inc.
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Priority to AU75516/94A priority Critical patent/AU7551694A/en
Publication of WO1995003833A1 publication Critical patent/WO1995003833A1/fr

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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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1137—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against enzymes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
    • C12Y207/11—Protein-serine/threonine kinases (2.7.11)
    • C12Y207/11013—Protein kinase C (2.7.11.13)
    • 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
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/31—Chemical structure of the backbone
    • C12N2310/318—Chemical structure of the backbone where the PO2 is completely replaced, e.g. MMI or formacetal
    • 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
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/35—Nature of the modification
    • C12N2310/351—Conjugate
    • C12N2310/3513—Protein; Peptide

Definitions

  • This invention is directed to compounds that are not polynucleotides yet which bind in a complementary fashion to DNA and RNA strands.
  • the invention concerns compounds wherein naturally-occurring nucleobases or other nucleobase-binding moieties are covalently bound to a polyamide backbone.
  • This invention is further directed to therapies, diagnostics, and research reagents for disease states which respond to modulation of the expression of protein kinase C. Palliation and therapeutic effect result.
  • PNAs Peptide Nucleic Acids
  • Oligodeoxyribonucleotides as long as 100 base pairs (bp) are routinely synthesized by solid phase methods using commercially available, fully automatic synthesis machines. The chemical synthesis of oligoribonucleotides (RNAs) , however, is far less routine. Oligoribonucleotides are also much less stable than oligodeoxyribonucleotides, a fact which has contributed to the more prevalent use of oligodeoxyribonucleotides in medical and biological research directed to, for example, gene therapy or the regulation of transcription or translation. Genes function by transferring information to a messenger RNA (mRNA) molecule, a process referred to as transcription.
  • mRNA messenger RNA
  • mRNA The interaction of mRNA with the ribosomal complex directs the synthesis of a protein encoded within its sequence. This synthetic process is known as translation and requires the presence of various co-factors and building blocks, the amino acids, and their transfer RNAs (tRNA) , all of which are present in normal cells.
  • tRNA transfer RNAs
  • RNA- synthesizing enzyme RNA polymerase
  • this recognition is preceded by sequence-specific binding of protein transcription factors to the promoter.
  • Other proteins which bind to the promoter, but whose .binding prohibits action of RNA polymerase, are known as repressors.
  • gene activation is typically regulated positively by transcription factors and negatively by repressors.
  • Most conventional drugs function by interaction with and modulation of one or more targeted endogenous proteins, e . g. , enzymes.
  • drugs are typically not specific for targeted proteins but interact with other proteins as well.
  • a relatively large dose of drug must be used to effectively modulate a targeted protein.
  • Typical daily doses of drugs are from 10 "5 -10 "1 millimoles per kilogram of body weight or 10 "3 -10 millimoles for a 100 kilogram person. If this modulation could instead be effected by interaction with and inactivation of mRNA, a dramatic reduction in the necessary amount of drug could likely be achieved, along with a corresponding reduction in adverse side effects. Further reductions could be achieved if such interaction could be rendered site-specific.
  • Oligodeoxynucleotides offer such opportunities.
  • synthetic oligodeoxynucleotides could be used as antisense probes to block and eventually lead to the breakdown of mRNA.
  • synthetic DNA could suppress translation in vivo.
  • it also may be possible to modulate the genome of an animal by, for example, triple helix formation using oligonucleotides or other DNA recognizing agents.
  • triple helix formation there are a number of drawbacks associated with triple helix formation. For example, it can only be used for homopurine sequences and it requires unphysiologically high ionic strength and low pH.
  • oligonucleotides are impractical both in the antisense approach and in the triple helix approach because they have short in vivo half-lives, and are difficult to prepare in more than milligram quantities and, thus, are prohibitively costly. They are also poor penetrators of the cell membrane.
  • peptide nucleic acids are synthesized by adaptation of certain peptide synthesis procedures, either in solution or on a solid phase.
  • the synthons used are certain monomer amino acids or their activated derivatives, protected by standard groups.
  • These oligonucleotide analogs also can be synthesized by using the corresponding diacids and diamines.
  • Peptide nucleic acid oligomers have been found to be superior to prior reagents in that they have significantly higher affinity for complementary single stranded DNA (ssDNA) .
  • ssDNA complementary single stranded DNA
  • These compounds are also able to form triple helices wherein a first PNA strand binds with RNA or ssDNA and a second PNA strand binds with the resulting double helix or with the first PNA strand.
  • PNAs generally possess no significant charge and are water soluble, which facilitates cellular uptake.
  • PNAs contain amides of non- biological amino acids, making them
  • PNAs can ideally be used to target RNA and ssDNA to produce antisense-type gene regulating moieties.
  • Reagents that bind sequence-specifically to dsDNA, RNA, or ssDNA have applications as gene targeted drugs useful for modulating metabolic processes such as metabolic regulatory dysfunctions, such as cancer.
  • PKC Protein Kinase C
  • PKC protein kinase C
  • DAGs diacylglycerols
  • PKC is the major, and perhaps only, cellular receptor through which a class of tumor-promoting agents called phorbol esters exert their pleiotropic effects on cells [Gescher et al. ,
  • Phorbols capable of tumor production can mimic the effect of DAG in activating PKC, suggesting that these tumor promotors act through PKC and that activation of this enzyme is at least partially responsible for the resulting tumorigenesis [Parker et al. , Science 233:853-866 (1986)].
  • Increased tumorigenicity is also correlated with overexpression of PKC in cultured cells inoculated into nude mice.
  • a mutant form of PKC induces highly malignant tumor cells with increased metastatic potential.
  • Sphingosine and related inhibitors of PKC activity have been shown to inhibit tumor cell growth and radiation-induced transformation in vivo [Endo et al. , Cancer Research 51:1613-1618 (1991); Borek et al., Proc. Natl . Acad. Sci . 88:1953-1957 (1991)].
  • a number of experimental or clinically useful anti-cancer drugs show modulatory effects on PKC. Therefore, inhibitors of PKC may be important cancer-preventive or therapeutic agents.
  • PKC has been suggested as a plausible target for more rational design of conventional anti-cancer drugs [Gescher, A. and Dale, I.L., Anti -Cancer Drug Design, 4:93-105 (1989)].
  • Psoriasis is characterized by inflammation, hyperproliferation of the epidermis and decreased differentiation of cells.
  • Various studies indicate a role for PKC in causing these symptoms.
  • PKC stimulation in cultured keratinocytes can be shown to cause hyperproliferation.
  • Inflammation can be induced by phorbol esters and is regulated by PKC.
  • DAG is implicated in the involvement of PKC in dermatological diseases, and is formed to an increased extent in psoriatic lesions.
  • Inhibitors of PKC have been shown to have both antiproliferative and antiinflammatory effects in vi tro .
  • Inhibition of PKC has been suggested as a therapeutic approach to the treatment of psoriasis [Hegemann, L. and G. Mahrle, Pharmacology of the Skin. H. Mukhtar, ed., p. 357-368, CRC Press, Boca Raton, FL, 1992] .
  • PKC is not a single enzyme, but a family of enzymes. At the present time at least seven isoforms
  • isozymes of PKC have been identified: isoforms ⁇ _, ⁇ , and ⁇ have been purified to homogeneity, and isoforms ⁇ , e, ⁇ and ⁇ have been identified by molecular cloning. These isozymes have distinct patterns of tissue and organ localization (see Nishizuka, Nature, 334:661-665 (1988) for review) and may serve different physiological functions. For example, PKC- ⁇ seems to be expressed only in the central nervous system. PKC-o; and - ⁇ are expressed in most tissues, but have different patterns of expression in different cell types. For example, both PKC-o; and PKC- ⁇ are expressed in, and have been purified from, human epidermis.
  • PKC- ⁇ has been detected mainly in keratinocytes of the basal layers of the epidermis
  • PKC- ⁇ is found mainly in the middle layers of the epidermis and Langerhans cells.
  • PKC-17 has been found predominantly in the skin and lungs, with levels of expression much higher in these tissues than in the brain. This is in contrast to other members of the PKC family which tend to be most abundantly expressed in the brain [Osada et al., J. Biol . Chem. 265:22434-22440 (1990)].
  • PKC isozymes listed here are preferred for targeting by the present invention, other isozymes of PKC are also comprehended by the present invention.
  • PKC isozymes may be involved in various disease processes depending on the organ or tissue in which they are expressed. For example, in psoriatic lesions there is an alteration in the ratio between PKC-o; and PKC- ⁇ , with preferential loss of PKC- ⁇ compared to normal skin [Hegemann, L. and G. Mahrle, Pharmacology of the Skin, H. Mukhtar, ed., p. 357-368, CRC Press, Boca Raton, FL, 1992] .
  • the present invention provides oligomers comprising peptide nucleic acids (PNAs) , that bind complementary ssDNA and RNA strands through their oligoribonucleotide ligands which are linked to a peptide backbone.
  • PNAs peptide nucleic acids
  • the sequence of the oligoribonucleotide ligands specifies the target to which they bind.
  • These PNA oligomers are useful as therapeutic agents for treating neoplastic, hyperproliferative, inflammatory and other disease states which respond to modulation of the expression of protein kinase C. These compositions are also useful in diagnostic applications and as research tools.
  • Oligomers of the present invention are comprised wherein at least one subunit of the oligomer is a peptide nucleic acid subunit of the formula:
  • L is one of the adenine, thymine, cytosine or guanine heterocyclic bases of the oligomer
  • C is (CR 6 R 7 ) y where R 6 is hydrogen and R 7 is selected from the group consisting of the side chains of naturally occurring alpha amino acids, or R 6 and R 7 are independently selected from the group consisting of hydrogen, (C 2 -C 6 )alkyl, aryl, aralkyl, heteroaryl, hydroxy, alkoxy, (C x - C 6 )alkylthio, NR 3 R 4 and SR 5 , where each of R 3 and R 4 is independently selected from the group consisting of hydrogen, (C x -C 4 )alkyl, hydroxy- or alkoxy- or alkylthio-substituted (C x -C 4 )alkyl, hydroxy, alkoxy, alkylthio and amino; and R 5 is hydrogen, (Ci-Cg)alkyl, hydroxy-, alkoxy-, or alkylthio- substituted (Ci-Cg)alkyl, or R 6 and R 7 taken together complete an
  • D is (CR 6 R 7 ) Z where R 6 and R 7 are as defined above; each of y and z is zero or an integer from 1 to 10, the sum y + z being greater than 2 but not more than 10;
  • G is -NR 3 CO-, -NR 3 CS-, -NR 3 SO- or -NR 3 S0 2 -, in either orientation, where R 3 is as defined above; each pair of A and B is selected such that:
  • A is a group of formula (Ila) , (lib) or (lie) and B is N or R 3 N + ; or
  • A is a group of formula (lid) and B is CH;
  • X is 0, S, Se, NR 3 , CH 2 or C(CH 3 ) 2 ;
  • Y is a single bond, 0, S or NR 4 ; each of p and q is zero or an integer from 1 to 5, the sum p+q being not more than 10; each of r and s is zero or an integer from 1 to 5, the sum r+s being not more than 10; each R 1 and R 2 is independently selected from the group consisting of hydrogen, (Ci-C 4 )alkyl which may be hydroxy- or alkoxy- or alkylthio- substituted, hydroxy, alkoxy, alkylthio, amino and halogen.
  • Subunits refers to basic unit which are chemically similar and which can form polymers. Repeating basic units form polymers referred to as "oligomers". Oligomers of the present invention may thus refer to oligomers in which substantially all subunits of the oligomer are subunits as described in Formula I. Oligomers of the present invention may also comprise one or more subunits which are naturally occuring nucleotides or nucleotide analogs as long as at least one subunit satisfies Formula I. Thus, oligomers as used herein may refer to a range of oligomers from oligomers comprising only one PNA subunit as defined in Formula I to oligomers in which every subunit is a PNA subunit as defined in Formula I.
  • Those subunits which are not PNA subunits comprise naturally occurring bases, sugars, and intersugar (backbone) linkages as well as non-naturally occurring portions which function similarly to naturally occurring portions.
  • Sequences of oligomers of the present invention are defined by reference to the L group (for PNA subunits) or nucleobase (for nucleotide subunits) at a given position.
  • the nomenclature is modeled after traditional nucleotide nomenclature, identifying each PNA subunit by the identity of its L group such as the heterocycles adenine (A) , thymine (T) , guanine (G) and cytosine (C) and identifying nucleotides or nucleosides by these same heterocycle residing on the sugar backbone.
  • L group such as the heterocycles adenine (A) , thymine (T) , guanine (G) and cytosine (C) and identifying nucleotides or nucleosides by these same heterocycle residing on the sugar backbone.
  • the sequences are conveniently provided in traditional 5' to 3' or amino to carboxy orientation.
  • Oligomers of the present invention may range in size from about 5 to about 50 subunits in length. In other embodiments of the present invention, oligomers may range in size from about 10 to about 30 subunits in length. In still other embodiments of the present invention oligomers may range in size from about 10 to about 25 subunits in length. In yet further embodiments of the present invention, oligomers may range in size from about 12 to about 20 subunits in length.
  • Established methods for the stepwise or fragmentwise solid-phase assembly of amino acids into peptides normally employ a beaded matrix of slightly cross- linked styrene-divinylbenzene copolymer, the cross-linked copolymer having been formed by the pearl polymerization of styrene monomer to which has been added a mixture of divinylbenzenes. A level of 1-2% cross-linking is usually employed. Such a matrix also can be used in solid-phase PNA synthesis in accordance with the present invention.
  • benzhydrylamino functionality (Pietta, et al . , J. Chem. Soc , 1970, 650) are the most widely applied. Regardless of its nature, the purpose of the functionality is normally to form an anchoring linkage between the copolymer solid support and the C-terminus of the first amino acid to be coupled to the solid support. As will be recognized, anchoring linkages also can be formed between the solid support and the amino acid N-terminus. It is generally convenient to express the "concentration" of a functional group in terms of millimoles per gram (mmol/g) . Other reactive functionalities which have been initially introduced include 4-methylbenzhydrylamino and 4-methoxybenzhydrylamino.
  • Preferred methods for PNA synthesis employ aminomethyl as the initial functionality, in that aminomethyl is particularly advantageous with respect to the incorporation of "spacer” or “handle” groups, owing to the reactivity of the amino group of the aminomethyl functionality with respect to the essentially quantitative formation of amide bonds to a carboxylic acid group at one end of the spacer-forming reagent.
  • a vast number of relevant spacer- or handle-forming bifunctional reagents have been described (see, Barany, et al . , Int . J. Peptide Protein Res . , 1987, 30, 705) , especially reagents which are reactive towards amino groups such as found in the aminomethyl function.
  • Representative bifunctional reagents include 4- (haloalkyl)aryl-lower alkanoic acids such as 4- (bromomethyl)phenylacetic acid, Boc-aminoacyl-4-
  • (oxymethyl)aryl-lower alkanoic acids such as Boc-aminoacyl-4- (oxymethyl)phenylacetic acid, N-Boc-p-acylbenzhydrylamines such as N-Boc-p-glutaroylbenzhydrylamine, N-Boc-4' -lower alkyl-p-acylbenzhydrylamines such as N-Boc-4' -methyl-p- glutaroylbenzhydrylamine, N-Boc-4' -lower alkoxy-p-acylbenz- hydrylamines such as N-Boc-4' -methoxy-p-glutaroyl-benzhy- drylamine, and 4-hydroxymethylphenoxyacetic acid.
  • One type of spacer group particularly relevant within the context of the present invention is the phenylacetamidomethyl (Pam) handle (Mitchell and Merrifield, J. Org. Chem. , 1976, 41 ,
  • the first amino acid to be coupled to the solid support can either be coupled to the free reactive end of a spacer group which has been bound to the initially introduced functionality (for example, an aminomethyl group) or can be reacted with the spacer- forming reagent.
  • the space-forming reagent is then reacted with the initially introduced functionality.
  • Other useful anchoring schemes include the "multidetachable" resins (Tam, et al . , Tetrahedron Lett . , 1979, 4935 and ⁇ J. Am. Chem. Soc , 1980, 102, 611; Tam, J. Org. Chem.
  • Suitable choices for N-protection are the tert- butyloxycarbonyl (Boc) group (Carpino, J. Am. Chem. Soc , 1957, 79, 4427; McKay, et al. , J. Am. Chem. Soc , 1957, 79, 4686; Anderson, et al. , J " . Am . Chem .
  • amino protecting groups are useful within the context of the present invention, but virtually any amino protecting group which largely fulfills the following requirements: (1) stability to mild acids (not significantly attacked by carboxyl groups) ; (2) stability to mild bases or nucleophiles (not significantly attacked by the amino group in question) ; (3) resistance to acylation (not significantly attacked by activated amino acids) . Additionally: (4) the protecting group must be close to quantitatively removable, without serious side reactions, and (5) the optical integrity, if any, of the incoming amino acid should preferably be highly preserved upon coupling.
  • side-chain protecting groups in general, depends on the choice of the amino protecting group, since the protection of side-chain functionalities must withstand the conditions of the repeated amino deprotection cycles. This is true whether the overall strategy for chemically assembling PNA molecules relies on, for example, differential acid stability of amino and side-chain protecting groups (such as is the case for the above- mentioned “Boc-benzyl” approach) or employs an orthogonal, that is, chemoselective, protection scheme (such as is the case for the above-mentioned "Fmoc-tBu” approach) ,
  • Novel monomer synthons may be selected from the group consisting of amino acids, diacids and diamines having general formulae:
  • the temporary protecting group, such as a Boc or Fmoc group, on the last-coupled amino acid is quantitatively removed by a suitable treatment, for example, by acidolysis, such as with trifluoroacetic acid, in the case of Boc, or by base treatment, such as with piperidine, in the case of Fmoc, so as to liberate the N-terminal amine function.
  • a suitable treatment for example, by acidolysis, such as with trifluoroacetic acid, in the case of Boc, or by base treatment, such as with piperidine, in the case of Fmoc, so as to liberate the N-terminal amine function.
  • the next desired N-protected amino acid is then coupled to the N-terminal of the last-coupled amino acid. This coupling of the C-terminal of an amino acid with the N- terminal of the last-coupled amino acid can be achieved in several ways.
  • the incoming amino acid in a form with the carboxyl group activated by any of several methods, including the initial formation of an active ester derivative such as a 2,4,5- trichlorophenyl ester (Pless, et al . , Helv. Chim. Acta, 1963, 46, 1609), a phthalimido ester (Nefkens, et al . , J. Am . Chem. Soc , 1961, 83 , 1263), a pentachlorophenyl ester (Kupryszewski, Rocz. Chem.
  • an active ester derivative such as a 2,4,5- trichlorophenyl ester (Pless, et al . , Helv. Chim. Acta, 1963, 46, 1609), a phthalimido ester (Nefkens, et al . , J. Am . Chem. Soc , 1961, 83 , 1263), a pent
  • the carboxyl group of the incoming amino acid can be reacted directly with the ⁇ -terminal of the last-coupled amino acid with the assistance of a condensation reagent such as, for example, dicyclohexylcarbodiimide (Sheehan, et al . , J. Am. Chem. Soc , 1955, 77, 1067) or derivatives thereof.
  • a condensation reagent such as, for example, dicyclohexylcarbodiimide (Sheehan, et al . , J. Am. Chem. Soc , 1955, 77, 1067) or derivatives thereof.
  • BOP Benzotriazolyl ⁇ - oxytrisdimethylaminophosphonium hexafluorophosphate
  • Castro's reagent see, e . g. , Rivaille, et al .
  • the next step will normally be deprotection of the amino acid moieties of the P ⁇ A chain and cleavage of the synthesized P ⁇ A from the solid support.
  • These processes can take place substantially simultaneously, thereby providing the free PNA molecule in the desired form.
  • stepwise chain building of achiral PNAs such as those based on aminoethylglycyl backbone units can start either from the N-terminus or the C-terminus, because the coupling reactions are free of racemization.
  • syntheses commencing at the C-terminus typically employ protected amine groups and free or activated acid groups
  • syntheses commencing at the N-terminus typically employ protected acid groups and free or activated amine groups.
  • PEPS polyethylene
  • PS pendant long-chain polystyrene
  • the loading capacity of the film is as high as that of a beaded matrix, but PEPS has the additional flexibility to suit multiple syntheses simultaneously.
  • the PEPS film is fashioned in the form of discrete, labeled sheets, each serving as an individual compartment. During all the identical steps of the synthetic cycles, the sheets are kept together in a single reaction vessel to permit concurrent preparation of a multitude of peptides at a rate close to that of a single peptide by conventional methods. It was reasoned that the PEPS film support, comprising linker or spacer groups adapted to the particular chemistry in question, should be particularly valuable in the synthesis of multiple PNA molecules, these being conceptually simple to synthesize since only four different reaction compartments are normally required, one for each of the four "pseudo- nucleotide" units.
  • the PEPS film support has been successfully tested in a number of PNA syntheses carried out in a parallel and substantially simultaneous fashion.
  • the yield and quality of the products obtained from PEPS were comparable to those obtained by using the traditional po ⁇ lystyrene beaded support.
  • experiments with other geometries of -the PEPS polymer such as, for example, non- woven felt, knitted net, sticks or microwellplates have not indicated any limitations of the synthetic efficacy.
  • solid supports which may be of relevance are: (1) Particles based upon copolymers of dimethylacrylamide cross-linked with N,N'- bisacryloylethylenediamine, including a known amount of N- tertbutoxycarbonyl-beta-alanyl-N' -acryloylhexamethylene- diamine.
  • spacer molecules are typically added via the beta alanyl group, followed thereafter by the amino acid residue subunits.
  • the beta alanyl-containing monomer can be replaced with an acryloyl sarcosine monomer during polymerization to form resin beads.
  • the polymerization is followed by reaction of the beads with ethylenediamine to form resin particles that contain primary amines as the covalently linked functionality.
  • the polyacrylamide-based supports are relatively more hydrophilic than are the polystyrene-based supports and are usually used with polar aprotic solvents including dimethylformamide, dimethylacetamide, N-methylpyrrolidone and the like (see Atherton, et al . , J. Am. Chem. Soc , 1975, 97, 6584, Bioorg. Chem. 1979, 8, 351), and J.C.S.
  • a second group of solid supports is based on silica-containing particles such as porous glass beads and silica gel.
  • silica-containing particles such as porous glass beads and silica gel.
  • One example is the reaction product of trichloro- [3- (4-chloro- methyl)phenyl]propylsilane and porous glass beads (see Parr and Grohmann, Angew. Chem. Internal . Ed. 1972, 11 , 314) sold under the trademark "PORASIL E” by Waters Associates,
  • Another exemplary composite contains a core of fluorinated ethylene polymer onto which has been grafted polystyrene (see Kent and Merrifield, Israel J. Chem. 1978, 17, 243) and van Rietschoten in "Peptides 1974" , Y. Wolman, Ed., Wiley and Sons, New York, 1975, pp. 113-116) ; and (4) contiguous solid supports other than PEPS, such as cotton sheets (Lebl and Eichler, Peptide Res . 1989, 2, 232) and hydroxypropylacrylate-coated polypropylene membranes (Daniels, et al . , Tetrahedron Lett . 1989, 4345), are suited for PNA synthesis as well.
  • solid- phase PNA synthesis in the context of the present invention is normally performed batchwise. However, most of the syn ⁇ theses may equally well be carried out in the continuous-flow mode, where the support is packed into columns (Bayer, et al . , Tetrahedron Lett . , 1970, 4503 and Scott, et al . , J. Chromatogr. Sci . , 1971, 9, 577) .
  • the rigid poly(dimethylacrylami- de) -Kieselguhr support Atherton, et al . , J. Chem. Soc Chem. Commun.
  • PNA molecules may be assembled enzymatically by enzymes such as proteases or derivatives thereof with novel specificities (obtained, for example, by artificial means such as protein engineering) .
  • PNA ligases for the condensation of a number of PNA fragments into very large PNA molecules; (6) since antibodies can be generated to virtually any molecule of interest, the recently developed catalytic antibodies (abzymes) , discovered simultaneously by the groups of Lerner (Tramantano, et al . , Science, 1986, 234 , 1566) and of Schultz (Pollack, et al . , Science, 1986, 234, 1570) , should also be considered as potential candidates for assembling PNA molecules.
  • Abzymes catalytic antibodies
  • Peptide nucleic acid oligomers hybridizable with, or targeted to genes or mRNA deriving from genes coding for PKC isozymes are provided by the present invention.
  • hybridizable is meant that at least 70% sequence homology is present.
  • peptide nucleic acid oligomers have at least 85% sequence homology to a desired target.
  • peptide nucleic acid oligomers of the present invention are at least 95% homologous to a target of interest.
  • Oligomers of the present invention comprising PNA subunits can be used in diagnostics, therapeutics and as research reagents and kits. Diagnostic and research reagents may be employed by contacting a cell or other biological sample such as blood, urine, cerebral fluid, ascites, etc. with oligomers of the present invention in vi tro .
  • oligomers of this invention hybridize to the PKC gene and its mRNA, sandwich and other assays can easily be constructed to exploit this fact. Furthermore, since the oligomers of this invention hybridize specifically to particular isozymes of the PKC mRNA, such assays can be devised for screening of cells and tissues for particular PKC isozymes. Such assays can be utilized for diagnosis of diseases associated with various PKC forms. Provision of means for detecting hybridization of oligomers with the PKC gene can routinely be accomplished. Such provision may include enzyme conjugation, radiolabelling or any other suitable detection systems. Kits for detecting the presence or absence of PKC may also be prepared.
  • Oligomers of the invention can be formulated in a pharmaceutical composition, which can include carriers, thickeners, diluents, buffers, preservatives, surface active agents and the like in addition to the oligomer.
  • Pharmaceutical compositions also can include one or more active ingredients such as antimicrobial agents, anti- inflammatory agents, anesthetics, and the like in addition to oligomer.
  • the pharmaceutical composition can be administered in a number of ways depending on whether local or systemic treatment is desired, and on the area to be treated. Administration can be topically (including opthalmically, vaginally, rectally, mtranasally) , orally, by inhalation, or parenterally, for example by intravenous drip, subcutaneous, intraperitoneal or intramuscular injection.
  • Formulations for topical administration can include ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders.
  • Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable.
  • Coated condoms may also be useful.
  • compositions for oral administration include powders or granules, suspensions or solutions in water or non-aqueous media, capsules, sachets, or tablets. Thickeners, flavorings, diluents, emulsifiers, dispersing aids or binders may be desirable.
  • Formulations for parenteral administration can include sterile aqueous solutions which also can contain buffers, diluents and other suitable additives. Dosing is dependent on severity and responsiveness of the condition to be treated, but will normally be one or more doses per day, with course of treatment lasting from several days to several months or until a cure is effected or a diminution of disease state is achieved. Persons of ordinary skill can easily determine optimum dosages, dosing methodologies and repetition rates. The following examples are provided for illustrative purposes only and are not intended to limit the invention.
  • PNA subunits for oligomers of the invention are prepared generally in accordance with the methods disclosed by WO 92/20702, incorporated by reference herein in its entirety.
  • Benzyhydrylamine resin (initially loaded 0.28 mmol/gm with Boc-L-Lys (2-chlorobenyloxycarbonyl) ) is swollen in DMF and an excess of a monomer to be coupled is added, followed by dicyclohexylcarbodiimide (0.15M in 50% DMF in dichloromethane) .
  • the Boc deprotection is accomplished by trifluoroacetic acid treatment.
  • the progress of the coupling reactions is monitored by quantitative ninhydrin analysis.
  • the PNA is released from the resin using anhydrous HF under standard conditions.
  • the products are purified using HPLC with acetonitrile-water (0.1%TFA) gradient and structure
  • PNA homopolymer has the structure:
  • the sequences, SEQ ID numbers and targets of these oligomers are shown in Table 1. TABLE 1
  • Oligomers targeted to the translation initiation codon (AUG) or 3' untranslated region (3' UTR) of PKC- ⁇ isoforms I or II were identified having specific sequences. These oligomers will be useful for the treatment of conditions modulated by or associated with PKC-/3 such as cell proliferation and differentiation. These sequences, SEQ ID numbers and targets of these oligomers are shown in Table 2. TABLE 2
  • Oligomers targeted to the translation initiation codon (AUG) or 5' untranslated region (5' UTR) of PKC- ⁇ were identified having specific sequences. These oligomers will be useful for the treatment of conditions modulated by or associated with PKC- ⁇ such as cell proliferation and differentiation.
  • the sequences, SEQ ID numbers and targets of these oligomers are shown in Table 3.
  • Oligomers targeted to the translation initiation codon (AUG) or 3' untranslated region (3' UTR) of PKC-77 were identified having specific sequences. These oligomers will be useful for the treatment of conditions modulated by or associated with PKC-17 such as cell proliferation and differentiation.
  • the sequences, SEQ ID numbers and targets of these oligomers are shown in Table 4.
  • Oligomers targeted to the translation initiation codon (AUG) , coding, stop or 3' untranslated region (3' UTR) of PKC-f were identified having specific sequences. These oligomers will be useful for the treatment of conditions modulated by or associated with PKC-f such as cell proliferation and differentiation.
  • the sequences, SEQ ID numbers and targets of these oligomers are shown in Table 5.
  • Oligomers targeted to the translation initiation codon (AUG) , coding or stop of PKC-e were identified having specific sequences. These oligomers will be useful for the treatment of conditions modulated by or associated with PKC-e such as cell proliferation and differentiation.
  • the sequences, SEQ ID numbers and targets of these oligomers are shown in Table 6.
  • Example 2 Cell culture and treatment with phorbol esters and oligomers targeted to PKC- ⁇ :
  • PKC protein half-lives have been reported to vary from 6.7 hours to over 24 hours [Young et al. , Biochem. J. 244:775-779 (1987); Ballester et al., J. Biol . Chem. 260:15194-15199 (1985)] . These long half-lives make inhibiting steady-state levels of PKC- ⁇ an unwieldy approach when screening PNA oligomers due to the long incubation times which would be required. We will therefore make use of the ability of phorbol esters to reversibly lower intracellular levels of PKC. Treatment of cells with phorbol esters causes an initial activation of kinase activity, followed by a down- regulation of PKC.
  • PKC-c ⁇ without affecting PKC-o; mRNA levels.
  • the basis of the assay to screen for potency of PNA oligomers targeted to PKC- o; is to initially lower PKC-o; protein levels by chronic treatment with PDBu, remove PDBu by extensively washing the cells (hence allowing the cells to synthesize fresh PKC-o; protein) , and incubate the cells with PNA oligomers intended to inhibit the resynthesis of new PKC- ⁇ protein.
  • A549 cells are grown to confluence in 6- well plates (Falcon Labware, Lincoln Park, NJ) in Dulbecco's modified Eagle's medium (DME) containing 1 g glucose/liter and 10% fetal calf serum (FCS, Irvine Scientific, Santa Ana,
  • DME Dulbecco's modified Eagle's medium
  • PNA oligomers of Tables 1 through 5 are added to a concentration of 1 ⁇ M and the cells are incubated for a further 4 hours at 37°C. Cells are washed once in 3 ml DME containing 0.1 mg/ml
  • PNA oligomers (1 ⁇ M) are added and the cells are incubated at
  • Cell extracts are electrophoresed on 10% SDS-PAGE mini-gels.
  • the resolved proteins are transferred to Immobilon-P membrane (Millipore, Bedford MA) by electrophoretic transfer and the membrane is blocked for 60 minutes in TBS (Tris-HCl pH 7.4, 150 mM NaCl) containing 5% nonfat milk.
  • TBS Tris-HCl pH 7.4, 150 mM NaCl
  • the membrane is then incubated for 16 hours at 4°C with monoclonal antibodies raised against PKC-o; (UBI, Lake Placid NY) diluted to 0.2 ⁇ g/ml in TBS containing 0.2% nonfat milk. This is followed by three washes in TBS plus 0.2% nonfat milk.
  • the membrane is then incubated for one hour with 125 i-labelled goat anti-mouse secondary antibody (ICN Radiochemicals, Irvine CA) .
  • ICN Radiochemicals 125 i-labelled goat anti-mouse secondary antibody
  • Membranes are then washed extensively in TBS plus 0.2% nonfat milk. Bands are visualized and quantitated using a Phosphorimager (Molecular Dynamics, Sunnyvale, CA) .
  • the peptide nucleic acid oligomer of SEQ ID NO. 2 was prepared using a peptide nucleic acid automatic synthesizer, the Millipore Expedite Nucleic Acid Synthesis System, Millipore Corporation, Bedford, MA. , under standard 1 ⁇ M synthesis conditions. Monomer, reagents and work-up were as follows: Monomers (0.2 M monomer, 0.2 M MDCHA and 0.3 M collidine)
  • Machine Cycle Each machine cycle included two 3 min deblock cycles, a 14 min couple step, a 5 min cap step and a 1 min piperidine wash following the cap.

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Abstract

L'invention concerne des oligomères qui permettent de moduler des processus métaboliques. Ces oligomères comprennent des sous-unités dont l'une au moins est une sous-unité d'acide nucléique de protéine. On décrit aussi des procédés thérapeutiques et diagnostiques.
PCT/US1994/008465 1993-07-29 1994-07-28 Oligomeres modulant la proteine kinase c WO1995003833A1 (fr)

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5744460A (en) * 1996-03-07 1998-04-28 Novartis Corporation Combination for treatment of proliferative diseases
EP0714449A4 (fr) * 1993-07-09 1998-12-02 Isis Pharmaceuticals Inc Modulation oligonucleotidique de la proteine kinase c
WO2000018781A1 (fr) 1998-09-29 2000-04-06 Isis Pharmaceuticals, Inc. Modulation anti-sens de l'expression de la survivine
US6190869B1 (en) 1999-10-26 2001-02-20 Isis Pharmaceuticals, Inc. Antisense inhibition of protein kinase C-theta expression

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
ANTISENSE RES. DEV., Volume 1, Number 1, issued 1991, FARESE et al., "Antisense DNA Downregulates Protein Kinase C Isozymes (beta and alpha) and Insulin-Stimulated 2-Deoxyglucose Uptake in Rat Adipocytes", pages 35-42. *
BIOCHEMISTRY, Volume 31, issued 1992, BAXTER et al., "PKCepsilon is Involved in Granulocyte-macrophage Colony-stimulating Factor Signal Transduction: Evidence From Microphysiometry and Antisense Oligonucleotide Experiments", pages 10950-10954. *
EXP. CELL. RES., Volume 205, issued March 1993, MAIER et al., "An Oligomer Targeted Against Protein Kinase Calpha Prevents Interleukin-1alpha Induction of Cyclooxygenase Expression in Human Endothelial Cells", pages 52-58. *
SCIENCE, Volume 252, issued December 1991, NIELSEN et al., "Sequence-selective Recognition of DNA by Strand Displacement With a Thymine-substituted Polyamide", pages 1497-1500. *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0714449A4 (fr) * 1993-07-09 1998-12-02 Isis Pharmaceuticals Inc Modulation oligonucleotidique de la proteine kinase c
US5744460A (en) * 1996-03-07 1998-04-28 Novartis Corporation Combination for treatment of proliferative diseases
WO2000018781A1 (fr) 1998-09-29 2000-04-06 Isis Pharmaceuticals, Inc. Modulation anti-sens de l'expression de la survivine
US6190869B1 (en) 1999-10-26 2001-02-20 Isis Pharmaceuticals, Inc. Antisense inhibition of protein kinase C-theta expression

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