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WO1995004068A1 - Oligomeres permettant de moduler le virus de l'immunodeficience humaine - Google Patents

Oligomeres permettant de moduler le virus de l'immunodeficience humaine Download PDF

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Publication number
WO1995004068A1
WO1995004068A1 PCT/US1994/008517 US9408517W WO9504068A1 WO 1995004068 A1 WO1995004068 A1 WO 1995004068A1 US 9408517 W US9408517 W US 9408517W WO 9504068 A1 WO9504068 A1 WO 9504068A1
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PCT/US1994/008517
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David J. Ecker
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Isis Pharmaceuticals, Inc.
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Priority to AU75164/94A priority Critical patent/AU7516494A/en
Publication of WO1995004068A1 publication Critical patent/WO1995004068A1/fr

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    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • 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
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16111—Human Immunodeficiency Virus, HIV concerning HIV env
    • C12N2740/16122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • 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
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16211—Human Immunodeficiency Virus, HIV concerning HIV gagpol
    • C12N2740/16222—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • 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
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16311—Human Immunodeficiency Virus, HIV concerning HIV regulatory proteins
    • C12N2740/16322—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

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. These compounds are useful for therapeutic and other applications directed to human immunodeficiency virus.
  • PNAs Peptide Nucleic Acids
  • DNAs 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) 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.
  • mRNA messenger RNA
  • tRNA transfer RNAs
  • RNA polymerase proteins which bind to the promoter, but whose binding prohibits action of RNA polymerase, are known as repressors. Thus, 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. However, such drugs are typically not specific for targeted proteins but interact with other proteins as well. Thus, 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.
  • Oligodeoxynucleotides offer such opportunities. For example, synthetic oligodeoxynucleotides could be used as antisense probes to block and eventually lead to the breakdown of mRNA. Thus, synthetic DNA could suppress translation in vivo .
  • triple helix formation 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.
  • PNAs denominated peptide nucleic acids
  • ligands are linked to a polyamide backbone through aza nitrogen atoms.
  • peptide nucleic acids are disclosed in which their recognition moieties are linked to the polyamide backbone additionally through amido and/or ureido tethers.
  • PCT/EP 92/01219 filed May 22, 1992 also discloses protein nucleic acids.
  • 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) . 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 biostable and resistant to enzymatic degradation, for example, by proteases.
  • 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 viral processes.
  • HIV Human Immunodeficiency Virus
  • HIV is the causative virus of AIDS.
  • a number of RNA secondary structures have recently been identified for which may serve as therapeutic targets. Some of these include the HIV TAR structures (Feng, S. and E.C. Holland, Nature, 1988, 334 , 165) , including the stem loops at nucleotide 1-59, and 60-104 according to the nucleotide sequence as described by Ratner et al . , Nature, 1985, 313 , 277; the boundary between the EGP/OMP regions of HIV (Le, S. et al . , Nucl . Acids Res . , 1988, 16, 5153; the boundary between the TMP/env genes of HIV (Le, S. et al . , Nucl .
  • Sarin and coworkers have also used chemically modified oligonucleotide analogs targeted to the cap and splice donor/acceptor sites.
  • Sarin, P.S. et al. Proc . Natl . Acad. Sci . USA, 1988, 85, 7448.
  • Zia and coworkers have also used an oligonucleotide analog targeted to a splice acceptor site to inhibit HIV. Zaia, J.A. et al . , J. Virol . , 1988, 62, 3914.
  • Matsukura and coworkers have synthesized oligonucleotide analogs targeted to the initiation of translation of the rev gene mRNA. Matsukura, M. et al .
  • 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 PNAs are extremely useful drugs for treating diseases like AIDS and other viral infections. 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 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, (C ⁇ Cg) 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 1 -C 4 ) alkyl, hydroxy- or alkoxy- or alkylthio-substituted (C- L - ) alkyl, hydroxy, alkoxy, alkylthio and amino; and R 5 is hydrogen, (C 1 -C 6 ) al
  • 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 S0- 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, (C- L -C J ) 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 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 occuring 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 nomeclature 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.
  • Solid- Phase Synthesis the principle of anchoring molecules onto a solid matrix, which helps in accounting for intermediate products during chemical transformations, is known as Solid- Phase Synthesis or Merrifield Synthesis (see, e . g. ,
  • 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.
  • 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.
  • 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 , 2015) which, deriving from the electron withdrawing effect of the 4-phenylacetamidomethyl group, is about 100 times more stable than the classical benzyl ester linkage towards the Boc-amino deprotection reagent trifluoroacetic acid (TFA) .
  • Certain functionalities e . g.
  • benzhydrylamino, 4- methylbenzhydrylamino and 4-methoxybenzhydrylamino which may be incorporated for the purpose of cleavage of a synthesized PNA chain from the solid support such that the C-terminal of the PNA chain is in amide form, require no introduction of a spacer group. Any such functionality may advantageously be employed in the context of the present invention.
  • spacer or handle groups An alternative strategy concerning the introduction of spacer or handle groups is the so-called "preformed handle” strategy (see, Tam, et al . , Synthesis, 1979, 955- 957) , which offers complete control over coupling of the first amino acid, and excludes the possibility of complications arising from the presence of undesired functional groups not related to the peptide or PNA synthesis.
  • spacer or handle groups of the same type as described above, are reacted with the first amino acid desired to be bound to the solid support, the amino acid being N-protected and optionally protected at the other side-chains which are not relevant with respect to the growth of the desired PNA chain.
  • 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. , 1985, 50, 5291), which provide more than one mode of release and thereby allow more flexibility in synthetic design.
  • 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. Soc , 1957, 79, 6180) normally in combination with benzyl-based groups for the protection of side chains, and the 9-fluorenylmethyloxy- carbonyl (Fmoc) group (Carpino, et al . , J. Am . Chem . Soc , 1970, 92, 5748 and J.
  • Boc tert- butyloxycarbonyl
  • amino protecting groups particularly those based on the widely-used urethane functionality, successfully prohibit racemization (mediated by tautomerization of the readily formed oxazolinone (azlactone) intermediates (Goodman, et al., J. Am. Chem. Soc , 1964, 86, 2918)) during the coupling of most ⁇ -amino acids.
  • racemization mediated by tautomerization of the readily formed oxazolinone (azlactone) intermediates (Goodman, et al., J. Am. Chem. Soc , 1964, 86, 2918)
  • oxazolinone azlactone
  • 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:
  • L, A, B, C and D are as defined above, except that any amino groups therein may be protected by amino protecting groups;
  • E is COOH, CSOH, SOOH, S0 2 OH or an activated derivative thereof; and
  • F is NHR 3 or NPgR 3 , where R 3 is as defined above and Pg is an amino protecting group.
  • 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.
  • it can be bound by providing 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 .
  • an active ester derivative such as a 2,4,5- trichlorophenyl ester (Pless, et al . , Helv. Chim. Acta, 1963, 46, 1609) , a phthalimido ester (
  • anhydride such as a symmetrical anhydride (Wieland, et al . , Angew. Chem. , Int . Ed. Engl . , 1971, 10, 336) .
  • 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.
  • 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 P ⁇ A 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.
  • This matrix is comprised of a polyethylene (PE) film with pendant long-chain polystyrene (PS) grafts (molecular weight on the order of IO 6 ) .
  • PE polyethylene
  • PS 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.
  • 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.
  • 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.
  • 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' - acryloylhexamethylenediamine.
  • 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 functionali ⁇ ty.
  • the polyacrylamide-based supports are relatively more hydrophilic than are the polystyrene-based supports and are usually used with polar aprotic solvents including dimethyl- formamide, dimethylacetamide, N-methylpyrrolidone and the like (see Atherton, et al . , J. Am . Chem. Soc , 1975, 97,
  • 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 trich- loro- [3- (4-chloromethyl) henyl]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, Framingham, MA, USA.
  • 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 hydroxypropyla- crylate-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). With respect to continuous- flow solid-phase synthesis, the rigid poly(dimethylacrylami- de) -Kieselguhr support (Atherton, et al . , J. Chem. Soc . Chem. Commun .
  • solid-phase technique is presently preferred in the context of PNA synthesis, other methodologies or combinations thereof, for example, in combination with the solid-phase technique, apply as well:
  • 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.
  • there has been considerable success in producing abzymes catalyzing acyl-transfer reactions see for example Shokat, et al .
  • Peptide nucleic acid oligomers hybridizable with, or targeted to, viral targets 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 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, intranasally) , 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. Such methodologies will be useful for targeting the following targets for treatment of viral manifestations.
  • HIV Human Immunodeficiency Virus
  • tat protein results in ncreased expression of other HIV genes up to 1,000 fold, including the tat gene itself. Because of this autoregulatory positive feedback, and the fact that the TAR sequence in included in the mRNA from every HIV transcript, a immense amount of viral gene expression is triggered when the tat gene is activated. The interaction between the tat gene and the TAR element is therefore crucial to the life cycle of the HIV, and specific disruption of this interaction is likely to interrupt the propagation of the virus.
  • HIV tat protein is responsible for triggering an enormous amount of viral gene expression, that this occurs by interaction with the TAR sequence which is incorporated into every HIV mRNA transcript, that the HIV TAR sequence functions as an RNA structure and that the correct TAR RNA structure is essential for tat transactivation.
  • a series of antiviral oligomers comprising PNA targeted to the translation initiation codon (AUG) , 3' untranslated region (3' UTR) , 5' splice junction (5' spljnct) , 3' splice junction (3' spljnct) , coding sequence, frameshift region or 5' untranslated region (5' UTR) of HIV gene selected from the group consisting of env, gag, pol, rev and tat are developed with specific oligomer sequences.
  • the oligomer sequences, SEQ ID numbers and targets of these oligomers are shown in Table 1. - 25 -
  • GTCTAACCAGAGAGACCC tat 61 CAGATCTGGTCTAACCAGAGACCC tat 62 GCTCCCAGGCTCAGATCT tat 63 GCCAGAGAGCTCCCAGGCTCAGATCT tat 64 GCCAGAGAGCTCCCAGGC tat 65 GCTTAAGCAGTGGGTTCCCT tat 66 CTTTATTGAGGCTTAAGCAG tat 67
  • Example 1 The following examples are provided for illustrative purposes only and are not intended to limit the invention.
  • Example 1 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.
  • 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 confirmed by fast atom bombardment mass spectrometry.
  • PNA homopolymer has the structure:
  • TAR and tat function has been studied by removing the genes from the HIV genome and studying them in cell lines in isolation.
  • Vectors have been constructed to study the interactions between the tat protein and TAR element.
  • the tat gene is expressed under the SV40 promoter.
  • the TAR region is expressed from a separate plasmid fused to an easily assayed reporter gene, the placental alkaline phosphatase gene (PAP) .
  • PAP placental alkaline phosphatase gene
  • Enzymatic activity in cell culture models has been shown to be dependent upon both the presence of the essential elements of the TAR region and the presence of the tat protein. Sharp, P.A. and R.
  • TAT/TAR trans activation can be conveniently assayed by placing the human PAP gene under the regulatory control of the HIV-1 LTR sequences, which contain enhancer, promoter, and tar elements.
  • a plasmid containing the HIV-1 LTR, pHIVCAT-0 (Feng, S. and E.C. Holland, Nature, 1988, 334 , 165) , contains HIV U3 in its entirety and R up through position +78 (a Hindlll site) . Digestion of this plasmid with a combination of Hindlll and Aatll releases the CAT cassette along with the SV40 sequences responsible for the processing of the RNA.
  • a second plasmid, pSV2Apap contains the PAP cassette with eukaryotic processing signals, under the transcriptional control of an SV40 promoter. Henthorn, P. et al . , Proc. Natl . Acad. Sci . USA, 1988, 85, 6342.
  • the PAP cassette and processing sequences are released from the plasmid by digestion with Hindlll and Aatll.
  • a new plasmid, pHIVPAP is created by ligating the Hindlll/Aatll fragment containing the HIV-1 LTR and vector sequences from pHIVCAT-0, to the Hindlll/Aatll PAP cassette from pSV2Apap.
  • Antiviral oligomers comprising PNA prepared in accordance with Example 1, having the following oligomer sequences: GGGGCGGGGCGGGGCGGGGCG (SEQ ID NO: 1) , GCGGGGTA (SEQ ID NO: 2), GGGGGGTA (SEQ ID NO: 3) , TCGGGGTT (SEQ ID NO: 7) , TTGGGGTT (SEQ ID NO: 8) , CCGGGGCC (SEQ ID NO: 9) , CGGGGGTA (SEQ ID NO: 10), TTGGGGTTGGGGTTGGGGTTGGGGG (SEQ ID NO: 13) , GGGGTTGGGG (SEQ ID NO: 14) , TTGGGGTTGGGGTTGGGGGG (SEQ ID NO: 15) , TTGGGGTTGGTTGGGGTT (SEQ ID NO: 16) , TTGGGGTTGGGGTTGGGGTTGGGG (SEQ ID NO: 17) , TTGGGGTTGGGGTT (SEQ ID NO: 18)
  • pcDEBtat and pHIVPAP are co-transfected into HeLa cells by calcium/phosphate precipitation.
  • the effects of the selected oligomers is determined as follows. HeLa cells are split 1:8 into 6-well dishes the day prior to the transfections. For each dish, 1 ug of pHIVPAP and 12ug of pcDEBtat are precipitated in 500 ⁇ l of HBS and 32 ⁇ l of 2.5 M CaCl 2 . The CaP0 4 precipitate is divided evenly between the 6 wells. Oligomers are precepitated in the same manner and added to the cells at different concentrations. The precipitate is allowed to sit on the cells for 20 minutes then complete media is added and the cells are incubated for an additional 4 hours. The cells are then shocked with 10% glycerol in HBS.
  • the cells are harvested in 0.5 ml of TBS, of which 0.1 mis are used for use in the protein assay. The remaining 0.4 mis of cell suspension is pelleted then resuspended in 50 ⁇ l TBS. Endogenous phosphatases are inactivated by heating the cells at 65°C for 30 min. The heat stable human placental alkaline phosphatase activity is assayed by the addition of PNPP (0.5 ml, 5 mM PNPP) to the cell suspension, which is then incubated at 37°C.
  • PNPP 0.5 ml, 5 mM PNPP
  • PAP Activity is determined at 30 minute intervals using 150 ⁇ l aliquots of the reaction mixture and measuring absorbance at 405 nm with a Titertek Multiscan MCC ⁇ 340 ELISA plate reader.
  • the PAP activity is normalized to the total protein in each well as determined by Bio-Rad protein assay, in which l ⁇ 5 of the harvested cells in TBS (0.1 ⁇ l) are added to 30 ⁇ l of Bio-Rad Protein Reagent, then incubated for 10 minutes at room temperature, followed by measurement of absorbance at 595 nm using the Titertek plate reader. It is expected that treatment of cells with oligomers directed to HIV will decrease or abrogate the production of PAP protein as a result of decreased HIV protein synthesis.
  • Example 3 Binding Properties of PNA compounds to HIV Sequence Targets
  • the binding properties of PNA compounds of the invention were studied by measuring the binding affinity of a 17 mer PNA oligomeric compound and its reverse 17 mer PNA oligomer (i.e. the same sequence synthesized in both amino to carboxy direction and carboxy to amino directions) .
  • the binding kinetics were measured by gel shift assay against both double stranded DNA and single stranded DNA.
  • Two 17 mer PNA compounds of the invention were synthesized as per the protocols of Example 1. These 17 mers PNA compounds, each complementary to the gag/pol region of the HIV genome, have the sequences: PNA Compound 1 gly-TTT TCC CTT CCT TTT CC-lys (SEQ ID NO.
  • PNA Compound 2 gly-CCT TTT CCT TCC CTT TT-gly-COOH (SEQ ID NO. 69) .
  • the double stranded DNA target was a 67 mer duplex containing the complementary sequence in opposite direction while the single stranded target was an exact complement 17 mer.
  • PNA compounds bound via a strand displacement mechanism against the double stranded DNA target .
  • Target strands were prepared for measuring binding in both the parallel direction (where the amino terminus of the PNA compound is aligned with the 5' terminus of the DNA compound) and the antiparallel direction (where the carboxyl terminus of the PNA compound is aligned with the 5' terminus of the DNA) .
  • Dissociation constants were determined by gel shift in a buffer containing 100 mM Na, 10 mM phosphate, 0.1 mM EDTA at pH 7.0.
  • the DNA target stands were 32 P end labeled. Binding affinity of the PNA compounds for the double stranded DNA target is shown in Table 1.
  • Binding affinity of the PNA compounds for the single stranded DNA target is shown in Table 2.

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Abstract

L'invention concerne une nouvelle classe de composés tels que des acides nucléiques peptidiques fixant des brins d'ADNss et d'ARN complémentaires plus efficacement qu'un ADN correspondant. Les acides nucléiques peptidiques comprennent généralement des ligands tels que des bases d'ADN existant à l'état naturel, liées à un squelette peptidique au moyen d'un segment de liaison approprié.
PCT/US1994/008517 1993-07-29 1994-07-28 Oligomeres permettant de moduler le virus de l'immunodeficience humaine WO1995004068A1 (fr)

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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996009832A1 (fr) * 1994-09-29 1996-04-04 Nauchno-Proizvodstvennoe Predpriyatie 'farmek' Hybride d'adn-arn et preparation d'adn, procedes d'obtention dudit hybride et de ladite preparation a partir de laitance d'esturgeon, et compose pharmaceutique a base dudit hybride d'adn-arn
US6015710A (en) * 1996-04-09 2000-01-18 The University Of Texas System Modulation of mammalian telomerase by peptide nucleic acids
US6063569A (en) * 1995-06-07 2000-05-16 Perseptive Biosystems, Inc. Methods for automated synthesis of PNA-DNA chimeras and compositions thereof
US6133444A (en) * 1993-12-22 2000-10-17 Perseptive Biosystems, Inc. Synthons for the synthesis and deprotection of peptide nucleic acids under mild conditions
EP1202750A1 (fr) * 1997-04-28 2002-05-08 BURCOGLU, Arsinur Traitement de l'infection par vih et de ses infections opportunistes
US6776986B1 (en) 1996-06-06 2004-08-17 Novartis Ag Inhibition of HIV-1 replication by antisense RNA expression
WO2004083426A1 (fr) * 2003-03-19 2004-09-30 Junsei Chemical Co. Ltd. Nouvel oligonucleotide antisens et agent anti-vih
EP2266616A3 (fr) * 2003-09-26 2011-09-14 Ganymed Pharmaceuticals AG Identification d'antigènes de surfaces cellulaires associés à des tumeurs pour le diagnostic et la thérapie

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
PROC. NATL. ACAD. SCI. U.S.A., Volume 85, issued August 1988, GOODCHILD et al., "Inhibition of Human Immunodeficiency Virus Replication by Antisense Oligodeoxynucleotides", pages 5507-5511. *
PROC. NATL. ACAD. SCI. U.S.A., Volume 85, issued October 1988, AGRAWAL et al., "Oligodeoxynucleotide Phosphoramidates and Phosphorothioates as Inhibitors of Human Immunodeficiency Virus", pages 7079-7083. *
PROC. NATL. ACAD. SCI. U.S.A., Volume 86, issued October 1989, AGRAWAL et al., "Inhibition of Human Immunodeficiency Virus in Early Infected and Chronically Infected Cells by Antisense Oligodeoxynucleotides and Their Phosphorothioate Analogues", pages 7790-7794. *

Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6133444A (en) * 1993-12-22 2000-10-17 Perseptive Biosystems, Inc. Synthons for the synthesis and deprotection of peptide nucleic acids under mild conditions
US6172226B1 (en) 1993-12-22 2001-01-09 Perseptive Biosystems, Inc. Synthons for the synthesis and deprotection of peptide nucleic acids under mild conditions
WO1996009832A1 (fr) * 1994-09-29 1996-04-04 Nauchno-Proizvodstvennoe Predpriyatie 'farmek' Hybride d'adn-arn et preparation d'adn, procedes d'obtention dudit hybride et de ladite preparation a partir de laitance d'esturgeon, et compose pharmaceutique a base dudit hybride d'adn-arn
US6063569A (en) * 1995-06-07 2000-05-16 Perseptive Biosystems, Inc. Methods for automated synthesis of PNA-DNA chimeras and compositions thereof
US6015710A (en) * 1996-04-09 2000-01-18 The University Of Texas System Modulation of mammalian telomerase by peptide nucleic acids
US6046307A (en) * 1996-04-09 2000-04-04 The University Of Texas System Modulation of mammalian telomerase by peptide nucleic acids
US6776986B1 (en) 1996-06-06 2004-08-17 Novartis Ag Inhibition of HIV-1 replication by antisense RNA expression
EP1202750A1 (fr) * 1997-04-28 2002-05-08 BURCOGLU, Arsinur Traitement de l'infection par vih et de ses infections opportunistes
EP1202750A4 (fr) * 1997-04-28 2002-10-16 Arsinur Burcoglu Traitement de l'infection par vih et de ses infections opportunistes
WO2004083426A1 (fr) * 2003-03-19 2004-09-30 Junsei Chemical Co. Ltd. Nouvel oligonucleotide antisens et agent anti-vih
EP2266616A3 (fr) * 2003-09-26 2011-09-14 Ganymed Pharmaceuticals AG Identification d'antigènes de surfaces cellulaires associés à des tumeurs pour le diagnostic et la thérapie
EP2332979A3 (fr) * 2003-09-26 2011-09-14 Ganymed Pharmaceuticals AG Identification d'antigènes de surfaces cellulaires associés à des tumeurs pour le diagnostic et la thérapie
EP2266615A3 (fr) * 2003-09-26 2011-09-21 Ganymed Pharmaceuticals AG Identification d'antigènes de surfaces cellulaires associés à des tumeurs pour le diagnostic et la thérapie

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