EP1356105A2 - Procede pour identifier des liaisons ou des structures initiales contre des motifs cibles arn et des interactions arn/proteine - Google Patents

Procede pour identifier des liaisons ou des structures initiales contre des motifs cibles arn et des interactions arn/proteine

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Publication number
EP1356105A2
EP1356105A2 EP01985820A EP01985820A EP1356105A2 EP 1356105 A2 EP1356105 A2 EP 1356105A2 EP 01985820 A EP01985820 A EP 01985820A EP 01985820 A EP01985820 A EP 01985820A EP 1356105 A2 EP1356105 A2 EP 1356105A2
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EP
European Patent Office
Prior art keywords
ribozyme
substrate
rna
polynucleotide
target molecule
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP01985820A
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German (de)
English (en)
Inventor
Andreas Jenne
Michael Blind
Michael Famulok
Seyed Hani Najafi-Shoushtari
Jörg HARTIG
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
NascaCell GmbH
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NascaCell GmbH
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Filing date
Publication date
Priority claimed from DE2000157853 external-priority patent/DE10057853A1/de
Priority claimed from DE2001144355 external-priority patent/DE10144355A1/de
Application filed by NascaCell GmbH filed Critical NascaCell GmbH
Publication of EP1356105A2 publication Critical patent/EP1356105A2/fr
Withdrawn legal-status Critical Current

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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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/4427—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems
    • A61K31/4439—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems containing a five-membered ring with nitrogen as a ring hetero atom, e.g. omeprazole
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
    • A61K31/7056—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing five-membered rings with nitrogen as a ring hetero atom
    • 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
    • C12N2310/10—Type of nucleic acid
    • C12N2310/11—Antisense
    • C12N2310/111—Antisense spanning the whole gene, or a large part of it
    • 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
    • C12N2310/10—Type of nucleic acid
    • C12N2310/12—Type of nucleic acid catalytic nucleic acids, e.g. ribozymes
    • C12N2310/121—Hammerhead

Definitions

  • the present invention relates to a method for identifying compounds (lead structures) which (a) bind specifically to a desired RNA target motif and can thereby inhibit or eliminate its function or which (b) have a desired RNA target motif Suppress associated connection and thereby inhibit or eliminate their function.
  • the compounds identified in this way serve as pharmaceutical lead substances for the production of pharmaceuticals, since they can specifically influence the cellular function of an RNA target motif or a compound associated with the RNA target motif.
  • the present invention further relates to a polynucleotide comprising a hammerhead ribozyme and an aptamer, a biosensor comprising this polynucleotide and a method for identifying a compound which binds to a target molecule, and the use of coumermycin, nosiheptide and patulin.
  • RNA molecules play a crucial role in the replication of retroviruses and thus in the manifestation of certain viral infections.
  • functional RNAs form an important class of therapeutic target molecules (“drug targets”) in pharmaceutical research [DS Eggleston, CD. Prescott & ND Pearson (ed.), The Many Faces of RNA, Academic Press (1998) 83-96; Y. Tor et al., Chem. Biol. 5 (1998), R277-R283; ND Pearson & CD. Prescott, Chem. Biol. 4 (1997), 409-414; T. Herrmann & E.
  • RNA-binding molecules have been used for combating bacterial infections for decades [WD Wilson & K. Li, Curr. Med. Chem. 7_ (2000), 73-98; Siegenthaler et al., Am. J. Med. 80 (1986), 2-14].
  • aminoglycosides such as neomycin B, paromomycin or streptomycin are able to inhibit bacterial translation and thus have an antibiotic effect [J.
  • RNA structures are outstandingly suitable as target molecules for active pharmaceutical ingredients for various reasons.
  • ribonucleic acids and deoxyribonucleic acids can form defined spatial structures and binding sites in order to specifically interact with other molecules [O.C. Uhlenbeck et al., Cell 90 (1997), 833-840; S. M. Pike, Bioorg. Med. Chem. 5 (1997), 1001-1248; C. S. Chow, Chem. Rev. 97 (1997), 1489-1513].
  • the risk of resistance formation in retroviruses against RNA therapeutics is also not very pronounced, since the RNA target molecules are highly conserved and initially unduplicated in the genome of the host cell [F.
  • RNAs as therapeutic target molecules to use drugs that target RNA structures are nowhere near as established as therapeutics that work at the DNA or protein level.
  • great efforts are therefore being made to develop antibiotics and antiviral agents that are directed against certain RNA motifs [DJ. Ecker & RH Griffey, Drug Disc. Today 4 (1999), 420-429].
  • RNA structure and RNA function databases it would be of the greatest value to identify as many small molecules as possible with specific binding properties for defined RNA structures [P. Brion & E. Westhof, Annu. Rev. Biophys. Biomol. Struct. 26: 113-137 (1997)].
  • RNA analysis such as gel retardation or filter binding experiments
  • kinetically stable RNA-ligand complexes for their detection and are therefore unsuitable for the screening of large compound libraries on an industrial scale.
  • Much more a robust, non-radioactive and widely applicable assay would be desirable, which enables the rapid, reliable identification of new RNA-binding molecules.
  • H.-Y. Mei et al. describe a method for the identification of inhibitors against the Group I self-splicing intron from Pneumocystis carinii [H.-Y. Mei et al. (1996) NAR 24: 5051-5053].
  • Group I intron splicing inhibitors are considered to be potential antibiotic agents in the fight against lung infections, which are triggered by the fungus Pneumocystis carinii and can be life-threatening for immunosuppressed patients.
  • the high-throughput screening carried out with the aid of the method described was able to identify new inhibitory lead structures from a compound library of approximately 300,000 low-molecular substances.
  • JE Arenas et al. have developed a screening method (“SCAN”) which enables the rapid identification of low-molecular ligands against different RNA target sequences [JE Arenas et al., Nucleic Acids Symp. Series 4_1 (1999), 13-16]. With the help of the method it was possible to isolate substances which bind to a certain regulatory RNA sequence, the so-called "epsilon-RNA" of the hepatitis B virus (HBV). Some of the isolated substances showed promising antiviral properties in a cell-based HBV Replication Model The method described by JE Arenas et al. Uses the different hybridization properties of free or ligand-bound RNA to complementary nucleic acid probes.
  • WO 98/18947A1 describes a method for characterizing and selecting RNA target molecules which bind to substances of therapeutic interest.
  • the method described is also suitable for the identification of new active substances with possible pharmacological activity.
  • RNA libraries are expressed in living cells and brought into contact with a substance to be tested.
  • the identification of an RNA target molecule pair is based on the phenotypic analysis of living cells, the method is not suitable for the rapid high-throughput screening of large substance libraries in vitro.
  • PCT / GB99 / 01761 describes a fluorescence-based method for identifying RNA-binding substances in vitro.
  • this method it is necessary to mark the RNA target structure to be examined and an already known RNA ligand of this target structure with a fluorophoric dye group. If the RNA and ligand are spatially separated from one another, the fluorescence of the fluorophoric groups can be measured. However, when a 1: 1 complex of RNA and ligand is formed, the fluorescence is quenched.
  • the identification of a new RNA-binding substance is based on the fact that the fluorescence quenching is canceled in the presence of a competitor competing for the RNA binding and a measurable signal is detected.
  • the method is suitable for high-throughput screening of substance libraries, it is not generally applicable, since in any case it requires knowledge of an RNA ligand which has already been identified.
  • the method should not have the disadvantages of the methods briefly discussed above in the prior art and should allow the construction of a robust, non-radioactive and automatable assay for the identification of substances with possible pharmacological activity which (a) bind directly to RNA structures or (b) disrupt the interaction between an RNA structure and an associated compound.
  • RNA construct used in the assay is referred to below as the target reporter construct (TRK).
  • TRK target reporter construct
  • RNA target motif If the RNA target motif is unbound, a signal with a certain intensity can be detected, as a result of a binding event (eg the binding of a low molecular weight substance to the target motif) the measured signal changes. As a result, it is possible to detect and quantify the binding of the substance to the RNA.
  • a binding event eg the binding of a low molecular weight substance to the target motif
  • RNA target motif e.g. to an RNA-binding protein
  • a signal with a certain intensity can also be detected. If the RNA-binding molecule is displaced from the binding site on the RNA, the measured signal changes and thus enables the displacement event to be detected. In this way, a substance with the desired binding properties for the original RNA-binding molecule can be identified.
  • the present invention thus relates to a method for identifying compounds which specifically bind to a desired RNA target motif and can thereby inhibit or eliminate its function, which is characterized by the following steps:
  • step (c) contacting the TRK from step (a) and the ribozyme substrate from step (b) with the compound which identifies, for example with a candidate from a substance library or a mixture containing this compound;
  • the present invention also relates to a method for identification of compounds which can displace a compound associated with a desired RNA target motif (for example a compound naturally associated with this RNA in the cell) and can thereby inhibit or eliminate its function.
  • This method is characterized by the following steps: Production of a construct (target reporter construct; TRK) from a reporter ribozyme domain (I) and the RNA target motif (II), where (I) and (II) by an RNA linker is connected to one another and the reporter ribozyme domain (I) changes its biological activity after displacement of the compound associated with the desired RNA target motif from the RNA target motif (II); Preparation of a signaling ribozyme substrate which bind specifically to the reporter ribozyme domain (I) and, preferably, can be cleaved by it; Contacting the TRK from step (a) with the compound associated with the RNA target motif;
  • step (c) Contacting the complex from step (c) and the ribozyme substrate from step (b) with the compound to be identified or a mixture containing this compound, for example with a candidate from a substance library; and determining the displacement of the compound associated with the RNA target motif, preferably based on the cleavage of the ribozyme substrate.
  • the step of producing the signaling ribozyme substrate described in the above methods can be omitted if a substrate is already known for the reporter ribozyme domain used in the TRK.
  • the step of providing a signaling ribozyme substrate and, if necessary, adding it to the process or the reaction mixture then takes its place.
  • reporter ribozyme domain refers to a ribozyme which is modified such that it can, for example, specifically cleave a suitable substrate RNA, which gives a measurable signal.
  • Ribozymes for example hammerhead ribozymes (HHR) are catalytic RNA molecules with the ability to sequence-specifically cleave other RNA molecules on phosphorus diester bonds.
  • the hammerhead ribozyme structure comprises three double-stranded regions (helices I, II, and III) which flank the cleavable phosphorus diester bond as well as two highly conserved single-stranded bonds Sequences [O. Uhlenbeck, Nature 328 (1987) 596- 600].
  • ribozymes which can split phosphodiester bonds into trans, ie intermolecularly, are suitable for the purposes of the invention.
  • ribonuclease P C Guerrier-Takada et al., Cell 44 (1983), 849-857
  • the known naturally occurring ribozymes hammerhead ribozyme, hairpin ribozyme, hepatitis delta virus ribozyme, neurospora mitochondrial VS ribozyme, group I and group II introns
  • self-cleaving or self-splicing catalysts which act in ice (intramolecular) [Review article in P.
  • ribozyme variants By separating the catalytic core sequence from a substrate sequence containing the cleavage site, ribozyme variants can thus be obtained (corresponding to the term “reporter ribozyme domain”) which can cleave almost any target RNA intermolecularly under physiological conditions [J. Haselhoff, W.
  • ribozyme The hydrolysis of the target sequence to be cleaved is always initiated by the formation of a catalytically active complex consisting of ribozyme and substrate RNA. After cleavage, the hydrolyzed substrate oligonucleotide dissociates from the ribozyme
  • Trans-cleaving ribozymes can be developed based on the ribozyme sequence by dividing the ribozyme into two areas, one of which is the cleavage site (substrate) and the other is the catalytic domain (in trans ribozyme) contains by experimental testing, ie measurement of the cleavage activity of different A ribozyme-substrate constructs, particularly active trans ribozymes can be determined.
  • ribozyme The catalytic activity of the ribozyme part thus gives a measurable signal which indicates the binding of a molecule to the RNA target domain or its detachment
  • ribozyme being both natural and modified ribozymes and DNA enzymes, so-called deoxyribozymes (R. Breaker, Chem. Rev. 97 (1997), 371-390; A. Jenne & M. Famulok, Top. Curr. Chem. 202 (1999).
  • ribozymes can also use other suitable ribozymes for signaling, for example ribozymes with RNA ligase activity
  • the binding event can be detected by PCR [MP Robertson & AD Ellington, Nat. Biotechnol. 17 (1999), 62-66].
  • the detection can preferably also be carried out by fluorescence measurement using so-called “Taq-man” probes (KJ Livak et al., PCR Methods Appl. 4 (1995), 357-362] ,
  • RNA target motif used here relates to RNA molecules or parts thereof which, due to their sequence or structure, fulfill a specific function within the cell.
  • the motifs can either occur naturally in the cell or be synthetic (eg intracellularly expressed RNA aptamers, so-called “intramers”, see below; M. Blind, et al., Proc. Natl. Acad. Sci. USA 96 (1999), 3603-3610).
  • the RNA target motif is identical to the reporter ribozyme, ie the ribozyme itself is the target.
  • therapeutically relevant ribozyme-controlled processes are self-splicing in pathogenic microorganisms, splicing in human cells (eg sickle cell anemia), tRNA processing by RNaseP, or RNA processing of the hepatitis delta virus genome [PC Turner (ed.), Methods in Molecular Biology: Ribozyme Protocols, Vol. 74 (1997), Humana Press, Totowa, NJ, USA].
  • An important class of RNA target motifs are structurally unique and highly conserved RNA structural elements, to which an important biological function can be assigned [AS Brodsky & JR Williamson, J. Mol.
  • RNA target motif should not be too long and preferably not exceed a length of 60 nucleotides, more preferably a length of 40 nucleotides.
  • RNA or DNA aptamers are of particular importance.
  • Aptamers are artificially selected nucleic acids with specific and sometimes high-affinity binding properties against a large number of different molecules [M. Famulok & A. Jenne, Curr. Opin. Chem. Biol. 2 (1998), 320-327; M. Famulok, Curr. Opin. Struc. Biol. 9_ (1999), 324-329; S.E. Osborne & A.Ellington. Chem.
  • Aptamers and their intracellular equivalents, the intramers are target-specific macromolecular lead substances that have important pharmacological properties exhibit later therapeutically active product. Aptamers are therefore outstandingly suitable for the development of low-molecular therapeutic agents, since the "therapeutic information" stored in the aptamer can be used by the method claimed here.
  • a measurable signal is generated in the methods according to the invention by the macromolecular aptamer being activated by another substance is displaced from the binding site on the target protein, so it can be assumed with high probability that the identified substance has properties similar to those of the aptamer (eg inhibition of the function of the target protein), in contrast to many other conventional screening Assays, the native target protein can be used without potentially disruptive modifications, such as dye or isotope labeling.
  • target reporter construct refers to the linkage of the reporter ribozyme domain and the RNA target motif via an RNA linker (see definition below), the linkage taking place in such a way that the reporter Ribozyme domain after specific binding of a specific ligand (the compound to be identified) to the RNA target motif, the reporter ribozyme domain changes, receives or loses its biologically active conformation.
  • RNA linker The person skilled in the art can use suitable techniques, meanwhile, to develop suitable target reporter Produce constructs (Soukup and Breaker, Current Opinions in Structural Biology 10 (2000), 318-325)
  • TRK in which the reporter ribozyme domain and the RNA target motif are identical, in particular omitted here also the presence of an RNA linker.
  • RNA molecules whose functions can be regulated by binding to proteins play an important role in many cellular processes [KJ Addess et al., J. Mol. Biol. 274 (1997), 72-83; MJ. Gait & J. Kam, Trends Biochem. Be. 18: 255-259 (1993)]. It has recently been shown that regulatable RNAs - in this case allosteric ribozymes - can be obtained by rational design or by in vitro selection. One made use of the fact that the spatial structure of a ribozyme was replaced by structural Changes can be stabilized or destabilized, and that major structural changes in most cases affect the catalytic activity of the ribozyme.
  • aptazyme has meanwhile become established in the literature for these allosterically regulatable ribozymes.
  • An aptazyme is characterized by two independent structural domains, namely a catalytically active RNA motif ("ribozyme”) and a ligand-binding RNA motif ( "Aptamer”), which represents the receptor domain.
  • ribozyme catalytically active RNA motif
  • Adamer ligand-binding RNA motif
  • Aptazymes are therefore suitable, for example, as molecular switches for the detection of small molecules or as controllable ones Units for the conditional control of gene expression [see for example WO 94/13791 and PCT 98/08974] This also applies to the oligonucleotides according to the invention.
  • RNA linker used here relates to an RNA sequence which connects the reporter ribozyme domain and the RNA target motif with one another in such a way that the signaling is conveyed via conformal changes in the RNA structure.
  • This “RNA Link” is of particular importance in the method according to the invention [GA Soukup & RR Breaker, Structure 7 (1999), 783-791; GA Soukup & RR Breaker, Trends Biotech. 17: 469-476 (1999)]. Suitable links can be selected either by empirical testing of various known sequences or by in vitro selection [GA Soukup & RR Breaker, PNAS USA 96 (1999) 3584-3589].
  • signaling ribozyme substrate used here relates to any RNA molecule which can specifically bind to the reporter ribozyme domain, from which, if it has its biologically active conformation, can be cleaved and also allows detection of the cleavage. This also presupposes that the cleaved ribozyme substrate is distinguishable from the uncleaved ribozyme substrate and an immediately measurable signal is generated.
  • the ribozyme substrate carries at one end an anchor group which allows its immobilization to a suitable matrix and at its other end a reporter group which serves to detect the immobilized (uncleaved) ribozyme substrate.
  • the ribozyme substrate In the case of inactive TRK (ie in the absence of a suitable ligand for the RNA target motif) the ribozyme substrate remains intact and can be easily detected after its immobilization on the matrix, since the anchor group is still connected to the reporter group.
  • TRK is active (ie after the ligand to be identified has been attached to the RNA target motif)
  • the reporter-specific signal cannot be detected, since the reporter group was separated from the anchor group as a result of the cleavage of the substrate.
  • the ribozyme substrate can also be immobilized via complementary sequence hybridization, provided the cleavage site and reporter group are located beyond the hybridization site. Easily detectable reporter groups that are easy to couple to nucleic acid ends are, for example, 32 P, dye molecules and molecules that can be detected with labeled antibodies.
  • the cleaved ribozyme substrate can also be replaced by a number of others Methods which are known to the person skilled in the art are detected and include, for example, gel electrophoresis and PCR.
  • the signaling ribozyme substrate is essentially complementary to the sequence (s) of the reporter ribozyme domain responsible for substrate binding, i.e. it has a complementarity that allows attachment to the ribozyme in a manner that ensures effective and specific cleavage of the ribozyme substrate.
  • the ribozyme substrate is preferably completely complementary to the sequences of the reporter ribozyme domain responsible for substrate binding.
  • the length of the attached region of the ribozyme substrate is preferably 8 to 14 nucleotides [P. Turner ed., Ribozyme protocols, Humana press (1997), 151-159, 253-264].
  • the ribozyme substrate can contain additional sequences at its 5 'and / or 3' end which are not involved in the attachment to the ribozyme.
  • the compounds or candidate compounds to be identified can in principle be any compound, which can belong to a wide variety of compound types, and the person skilled in the art is also familiar with a large number of sources which contain compounds suitable for the screening method according to the invention.
  • substance libraries including antisense nucleic acids; however, libraries with low molecular weight molecules that meet certain requirements are preferred, for example with regard to their low toxicity [DJ. Ecker & R.H. Griffey, Drug Disc. Today 4 (1999), 420-429].
  • constructs required for the method according to the invention are preferably produced in large amounts by in vitro transcription of the corresponding DNA sequences.
  • these DNA sequences are inserted into a vector which allows the inserted DNA to be multiplied in a suitable host, under the control of a suitable promoter, preferably the T7 promoter.
  • suitable vectors for propagation in prokaryotic or eukaryotic systems are, for example, pBR322, pNEB193, pUC18, pUC19 (Biolabs, USA.) [J. Sampson and O. Uhlenbeck, Proc. Natl. Acad. Be.
  • the plasmids are then isolated, purified and the in vitro transcription is carried out according to standard procedures.
  • the constructs used for the method according to the invention can also be produced by automated solid phase synthesis according to standard methods.
  • the reporter ribozyme domain comes from a hammerhead ribozyme.
  • hammerhead ribozymes and the production of intermolecularly cleaving variants reference is made to the above statements.
  • the above or the signaling ribozyme substrate is labeled twice, the cleaved substrate being easily distinguishable from the intact substrate.
  • a terminally biotinylated ribozyme substrate labeled with fluorescein at its other end can be used.
  • incubation is then carried out with a streptavidin-coated solid phase (e.g. with a commercially available microtiter plate) in order to enable the coupling of the biotinylated substrate end to the streptavidin matrix. After washing the matrix, it is measured.
  • reporter ribozyme domain In the presence of a ligand that specifically binds to the RNA target motif (reporter ribozyme domain is activated), no fluorescein-specific fluorescence or only a weak non-specific background fluorescence can be measured, since the fluorescein-labeled cleavage piece could not be immobilized.
  • the reporter-ribozyme construct was not activated due to the lack of a ligand that specifically binds to the RNA target motif, the proportion of uncleaved, immobilized ribozyme substrate can be quantified by measuring the fluorescein-specific fluorescence.
  • the double-labeled ribozyme substrate contains a fluorophoric group and a fluorescence-quenching group, wherein after cleavage by the reporter-ribozyme domain, the quenching of the fluorescence of the fluorophore by the fluorescence-quenching group is prevented.
  • FRET oligonucleotides are described, for example, in KJ Livak, SJA Flood, J. Marmaro, W. Giusti, K. Deetz, PCR Meth. Appln 4: 357-362 (1995).
  • FAM 6-carboxy-fluorescein
  • TET tetrachloro-6-carboxy-fluorescein
  • HEX hex
  • the cleavage pieces can separate from one another in solution: the fluorescence of the fluorophore is no longer quenched intramolecularly. If a suitable ligand is attached to the RNA target motif, which leads to a biologically active reporter ribozyme domain, this can be determined by generating a measurable fluorescence signal by cleaving the substrate.
  • the method according to the invention is particularly suitable for the industrial high-throughput screening of substance libraries, since it is simple to carry out and can be easily adapted to different microtiter plate formats [X. Chen et al., 1998 Genome Res. 8: 549-556; KP Bjornson et al., 1994 Biochemistry 33: 14306-14316; AR Gelsthorpe et al., Tissue Antigens 54 1999), 603-614; JE Gonzalez et al., Drug Discov. Today 4 (1999), 431-439]. In particular, radioactive waste is avoided, which would otherwise have to be disposed of at high cost.
  • This embodiment of the method according to the invention also has the advantage of detecting the binding of a ligand very sensitively, since the catalytic cleavage of the FRET substrate leads to a significant signal amplification [with regard to the determination of the cleavage activity of hammerhead ribozymes in a very short time by fluorescence measurement in the Microtiter plate format see also Jenne et al., Angew. Chem. Hl (1999), 1383-1386.].
  • RNA linker that connects the RNA target sequence with the ribozyme domain.
  • Methods for labeling ribonucleic acids with fluorophoric or fluorescence-quenching groups and techniques for measuring energy transfer (quenching) have already been described in detail [Turner (ed.), Ribozyme protocols, Humana press (1997), 241-251].
  • ⁇ '-Fluorophore and 3'-quencher-labeled RNA oligonucleotides are commercially available (e.g. 5'-FAM and 3'-TAMRA-labeled RNA from Eurogentec, Belgium).
  • the labeling is advantageously carried out at the RNA ends in order not to influence the hybridization to the reporter ribozyme domain.
  • nuclease-resistant ribozyme substrates In order to avoid the fluorescence emission associated with undesired cleavage (for example by nucleases in the transcription system), the use of nuclease-resistant ribozyme substrates is particularly advantageous (Eaton and Pieken, Annu. Rev. Biochem. 64 (1995), 837-863 and Shimayama et al., Nucleic Acids Res. 21 (1993), 2605-2611). This is particularly advantageous with regard to in vivo applications in which the double-labeled substrate is introduced exogenously into cells by suitable techniques (for example microinjection, liposome transport, etc.) (P. Turner (ed.), Ribozyme protocols, Humana press (1997), 417-451).
  • the double-labeled substrates are modified RNA oligonucleotides.
  • the substrate can contain deoxyribonucleotides and / or modified bases or / and 2'-modified ribose units. This increases the stability of the substrate in the cell extract (N. Taylor et al., Nucleic Acids Res. 20 (1992), 4559-4565).
  • the use of internally-labeled, instead of end-labeled oligonucleotide substrates can also contribute to an improved signal-to-noise ratio, since the fluorescence quenching is increased, inter alia, with shorter distances between the two groups (fluorophore and quencher).
  • the present invention also relates to a medicament which contains a compound identified by the method according to the invention. This also includes a compound derived therefrom, which can also bind to the RNA target motif, this compound containing, for example, only the portion or partial sequence of the originally identified compound or a portion or partial sequence which deviates therefrom and whose affinity for the RNA Target motif changed compared to the original connection, preferably increased.
  • the medicament is preferably combined with a suitable carrier.
  • Suitable carriers and the formulation of such medicaments are known to the person skilled in the art.
  • Suitable carriers include, for example, phosphate-buffered saline solutions, water, emulsions, for example oil / water emulsions, wetting agents, sterile solutions, etc.
  • the medicaments can be administered orally or parenterally.
  • Methods for parenteral administration include topical, intra-arterial, intramuscular, subcutaneous, intramedullary, intrathecal, intraventricular, intravenous, intraperitoneal, or intranasal administration.
  • the present invention relates to a kit (or assay) for carrying out the methods according to the invention, the kit comprising the following compounds described above: (a) a target reporter construct (TRK) and (b) a signaling ribozyme substrate.
  • the invention relates to a polynucleotide comprising a hammerhead ribozyme and an aptamer which is specific for a target molecule, the aptamer part of the polynucleotide preventing the formation of the catalytically active ribozyme by base pairing with sequences of the hammerhead ribozyme. This is achieved in that the sequence which causes the catalytic activity of the hammerhead ribozyme or a part thereof is no longer available by base pairing with the aptamer sequence for the formation of the base pairings required for the catalytic activity.
  • the invention relates to a polynucleotide comprising a hammerhead ribozyme and an aptamer, the aptamer being specific for a target molecule, in particular a polynucleotide according to the above aspect of Invention, which further comprises the bound target molecule specific for the aptamer and, as a result of the binding of the target molecule to the aptamer sequence, the enzymatic activity of the ribozyme is formed.
  • the invention relates to a polynucleotide comprising a hammerhead ribozyme and an aptamer for a target molecule, wherein the binding site of the catalytic domain of the ribozyme for a ribozyme substrate is blocked for binding of a substrate of the ribozyme in the absence of the target molecule of the aptamer.
  • the invention relates to a polynucleotide comprising a hammerhead ribozyme and an aptamer for a target molecule, in particular one according to one of the other aspects of the present invention, the binding site of the catalytic domain of the ribozyme for a ribozyme substrate in the presence of the target molecule of the aptamer is accessible for binding a substrate of the ribozyme.
  • the invention also relates in one aspect to a polynucleotide comprising a hammerhead ribozyme and an aptamer for a target molecule, in particular a polynucleotide according to one of the other aspects of the present invention, the base pairing pattern of the polynucleotide differing from that of the target molecule when the target molecule binds to the aptamer Polynucleotide in the absence of the target molecule from the aptamer, especially when the target molecule is not bound to the aptamer.
  • the present invention also relates to a polynucleotide which comprises a hammerhead ribozyme and an aptamer specific for a target molecule, both of which, ie hammerhead ribozyme and aptamer, are linked to one another and are dependent on the presence of the one specific for the aptamer
  • Different “base pair hybridization patterns” result in the target molecule in the ribozyme, which has the following effects:
  • a catalytically active ribozyme is formed in the polynucleotide according to the invention polynucleotide according to the invention.
  • the target molecule cannot bind to the aptamer sequence if there is a binding partner for the target molecule, for example an inhibitor, which prevents a specific interaction between the aptamer-nucleic acid part of the polynucleotide according to the invention and the target molecule.
  • a binding partner for the target molecule for example an inhibitor, which prevents a specific interaction between the aptamer-nucleic acid part of the polynucleotide according to the invention and the target molecule.
  • the polynucleotides described above are therefore the two states of a polynucleotide which are defined by the binding or non-binding of the target molecule of the aptamer.
  • the two states differ not only by their different secondary and tertiary structures, but also and in particular also by the type and, if appropriate, number of base pairings in the polynucleotide. In other words, the two states differ in their base pairing pattern.
  • This change in the base pairing pattern distinguishes the polynucleotides according to the invention from the ribozymes known in the art, including the allosteric ribozymes, and the aptazymes, in which the allosteric effect of the binding of the allosteric effector, such as for example the target molecule of the aptamer, changes only in one change Expresses secondary and / or tertiary structure, but not in a change in the base pairing pattern.
  • the oligonucleotides according to the invention represent target receptor constructs, the hammerhead ribozyme is a receptor ribozyme domain, and the aptamer is an RNA target motif in the sense of the present disclosure. It is within the scope of the present invention that the aptamer also is made up of deoxyribonucleotides.
  • the hammerhead ribozymes described herein are typically those consisting of three helices, two of which are formed by hybridization with the complementary sequence of their "target RNA" and the third is formed by a double strand within the ribozyme
  • the catalytic domain of the ribozyme consists of nucleotides, which are arranged between the double-stranded structures, and if one speaks of target RNA here, this refers to the RNA substrates against which Hammerhead ribozymes are cleaved in ice or in trans can.
  • the target RNA is typically one which represents a substrate, in particular a signal-generating substrate in the sense of the present disclosure, for the respective ribozyme.
  • polynucleotides according to the invention comprise a substrate for the catalytic activity of the ribozyme.
  • substrates have already been described in connection with the above aspects of the invention, in particular the configuration of the substrate as an FRET substrate. In this regard, reference is made to the corresponding disclosure herein.
  • the polynucleotide according to the invention has a sequence which is selected from the group SEQ ID No. 51 and SEQ ID No. 52 includes.
  • the polynucleotide according to the invention can preferably be designed as RNA. It is within the scope of the present invention that at least regions of the polynucleotide according to the invention are also designed as DNA, in particular those regions which are for pairing with the substrate, but also those which are involved in intramolecular base pairings.
  • An example of a substrate for the polynucleotides according to the invention is provided by SEQ ID No. 53 nucleic acid disclosed.
  • the structure on which the polynueleotides according to the invention are based is associated with surprising advantages over the allosteric ribozymes known in the prior art.
  • the most important of these advantages can be seen in the fact that the polynucleotides according to the invention make it possible to generate an allosteric ribozyme, the allosteric effector, ie the target molecule which binds to the aptamer part of the polynucleotide, being able to be selected practically as desired without the functionality of the Allosteric ribozyme undergoes impairment.
  • the polynucleotide according to the invention differs from the aptazymes described in the prior art, in which a so-called communication module is interposed between the receptor part or domain, ie the part of the allosteric ribozyme that binds the target molecule, and the catalytically active ribozyme domain, which is also included herein is referred to as an RNA linker.
  • a so-called communication module is interposed between the receptor part or domain, ie the part of the allosteric ribozyme that binds the target molecule
  • the catalytically active ribozyme domain which is also included herein is referred to as an RNA linker.
  • polynucleotides according to the invention are practically free from these types of restrictions.
  • the polynucleotides according to the invention are namely standardized or easily standardizable allosteric ribozymes or target-receptor constructs, the specificity of which can be set surprisingly easily to any target molecule against which an aptamer can be produced.
  • the reason for this flexibility in the specificity of the oligonucleotides according to the invention, ie the allosteric ribozyme according to the invention, is due to the structure of the hammerhead ribozymes. Due to the need to form three helices, it is possible to form at least one helix so that it is complementary to the nucleic acid sequence of the aptamer part of the allosteric ribozyme.
  • any aptamer can thus be inserted into the allosteric ribozyme, with the result that, by binding the target molecule of the aptamer to the aptamer part of the allosteric ribozyme, the necessary for the catalytic activity of the ribozyme Helices or non-helically present nucleotides are present as such and not base-paired with at least part of the aptamer sequence.
  • the allosteric ribozyme is only catalytically active in the target-binding form.
  • the present invention relates to a biosensor comprising a polynucleotide according to the invention, it being provided in a preferred embodiment that the polynucleotide is bound to a solid support.
  • biosensors also apply to the embodiments according to the invention.
  • the invention relates to a method for identifying a compound that binds to a target molecule, comprising the following steps:
  • candidate compound is used to refer to a compound that is used in these methods and that is being investigated to determine whether it binds to the target molecule, and particularly at a site recognized by the target molecule-specific aptamer.
  • Candidate compounds of this type are basically suitable as pharmaceutical lead substances if they influence the interaction between the aptamer and the target molecule.
  • a candidate connection is in particular also a “connection to be identified” in the sense of the present disclosure.
  • the candidate compound itself has already been tested beforehand with regard to its suitability for influencing the interaction between the aptamer and the target molecule.
  • This can happen, for example, in that an allosteric ribozyme according to the invention or a Oligonucleotide according to the invention is used, which comprises an aptamer, which binds to the target molecule but possibly to another site and thus allows a narrowing of the site at which there is an interaction between the aptamer and the target molecule.
  • the allosteric ribozymes used, more specifically aptazymes, differ in terms of specificity and / or affinity for their respective target molecule or parts thereof.
  • the candidate compound after it has been identified or validated by the method according to the invention, is subjected to a further identification or validation step, wherein what has been said above applies here and a correspondingly designed method according to the invention is used.
  • a further identification or validation step such a method can also be used, as is shown, for example, in FIG. 8a.
  • An allosteric ribozyme is provided which comprises an aptamer which is specific for the target molecule, as is also used in the process according to the invention, in which the base pairing pattern changes as a result of the binding of the target molecule.
  • the catalytic activity of the ribozyme portion of the allosteric ribozyme is determined in advance and then the target molecule is added to the reaction mixture according to the invention, the target molecule interacting with the aptamer domain binding the target molecule in the polynucleotide.
  • the catalytic activity of the allosteric ribozyme can optionally be determined in the presence of the target molecule before the candidate compound is subsequently added.
  • the substrate for the catalytic activity of the polynucleotide is finally provided and the substrate is added to the reaction mixture comprising the above-mentioned components.
  • the allosteric ribozyme can be one which comprises a hammerhead ribozyme part and an aptamer part, optionally connected to one another by a linker, for example an RNA linker, as described here, wpbei the aptamer is directed against a molecule that has a molecular weight greater than 300 Da, preferably greater than 1000 Da, more preferably greater than 2kDa, and more preferably greater than 5 kDa.
  • the target molecule is a peptide or protein. Such a protein-binding allosteric ribozyme according to the invention is described in the examples.
  • the method according to the invention in particular with the one that uses allosteric ribozymes that bind to the peptide or protein target molecule, it is possible for the first time in an inhibition assay to identify those compounds that interact with peptides or proteins.
  • the provision of the allosteric ribozymes according to the invention and their use in a method according to the invention for identifying compounds provides for the first time the possibility of determining lead structures which are associated with this highly relevant group of target molecules bind and thus can be used either directly or indirectly in the development of pharmaceutically active substances.
  • the two methods shown in FIG. 8 for the identification of compounds which interact with a target molecule - specifically - are coupled to one another, ie the compound already identified or validated in the method shown in FIG. 8 a is identified or validated again in the method shown in FIG. 8b.
  • the advantage of coupling the two methods is that the disadvantages inherent in the two methods, consisting of false positive or false negative results, can be compensated for.
  • false positive results can result from the fact that the candidate compound has a direct influence on the catalytic activity of the ribozyme, for example by binding to the ribozyme part of the aptazyme, and the effect observed in the experiment is not due to the fact that the candidate compound interacts with the target molecule.
  • the polynucleotide according to the invention is used in one of the two methods, in which a change in the base pair hybridization pattern occurs when the target molecule binds, and the easy adaptability associated with this type of polynucleotide different target molecules, a particularly reliable identification of compounds that react specifically with the target molecule can take place by coupling the two methods.
  • the candidate connection is subjected to a method which comprises the steps before step f) or after step g) of the method according to the invention:
  • the determination of the binding of the candidate compound to the target molecule is carried out by determining the catalytic activity of the ribozyme.
  • the catalytic activity of the ribozyme can be determined using the FRET substrate, as mentioned at the beginning.
  • the substrate contains a fluorophore and a fluorescence-quenching group and, after cleavage of the substrate by the catalytic activity of the ribozyme or the catalytic domain of the polynucleotide according to the invention, the quenching of the fluorescence is prevented or at least reduced.
  • the fluorophoric group is 6-carboxy-fluorescine and the fluorescent group 6-carboxy-tetramethylrhodamine or "Cy 3 ".
  • the present invention relates to a medicament which comprises a compound identified by the method according to the invention.
  • the invention relates to a kit for carrying out the method according to the invention
  • the present invention relates to the use of coumermycin for the manufacture of a medicament for the treatment of HIV and / or FIV.
  • the invention relates to the use of nosiheptide for the manufacture of a medicament for the treatment of HIV and / or FIV.
  • the invention relates to the use of patulin for the manufacture of a medicament for the treatment of HIV and / or FIV.
  • the present invention relates to the use of coumermycin, nosiheptide or patulin for the treatment of HIV and / or FIV.
  • the invention relates to the use of the compounds identified or validated according to the invention, e.g. B of coumermycin, nosiheptide or patulin, for the development of pharmaceutically active compounds that can ultimately be used as medicines.
  • the identified compounds e.g. B of coumermycin, nosiheptide or patulin.
  • the identified compounds can be used to produce derivatives which are suitable for their pharmacological properties can be optimized. Such properties include effectiveness, side effects, toxicity and the like.
  • the ribozyme substrate for the catalytic activity of the ribozyme is covalently bound to the ribozyme or the polynucleotide comprising the aptamer according to one of the aspects of the present invention and thus the catalytic reaction of the ribozyme is an intramolecular reaction.
  • the bond by means of which the ribozyme substrate and ribozyme are connected to one another is typically an intemucleosidic bond which can be present at either of the two ends of the ribozyme or the ribozyme substrate as well as within that which forms the ribozyme or the ribozyme substrate nucleic acid sequence.
  • Figure 1 Secondary structures of construct TRK1 and substrate SK1.
  • FIG. 2 Principle of fluorescence measurement: A so-called FRET oligonucleotide (SK1) was selected as the substrate for the reporter ribozyme TRK-1.
  • FAM fluorophoric group
  • TAMRA fluorescence quenching molecule
  • the fluorescence of the fluorophore is no longer quenched intramolecularly, and the ribozyme is available for the next catalytic cycle.
  • the elimination of the FRET effect causes a measurable increase in the FAM-specific fluorescence in the sample. Since the increase in fluorescence is directly proportional to the cleavage activity of the ribozyme, the reaction rates can be determined from the measured data.
  • Figure 4 Fluorescence measurements with TRK1 / SL1 which were obtained when screening the substance library. Time-dependent increase in the fluorescence signal for (a) the uninhibited TRK1 reaction, (b) the TRK1 reaction in the presence of substance # 332 and (c) the TRK1 reaction in the presence of substance # 425.
  • the measuring points and regression lines for the uncorrected reaction course, the corresponding control approach without TRK1 and the corrected reaction are shown in each case.
  • FIG. 5 Principle of the screening assay.
  • Each microtiter plate contains (i) the target protein to be investigated (ii) a substance from the compound library (iii) and a suitable reporter-ribozyme construct, for example an intramer-ribozyme construct (see application text). Fluorescence measurement can identify substances that are able to bind to the target protein to be examined. As a result, the RNA-ribozyme construct is displaced by the protein-binding domain, which leads to a measurable change in the Fluorescence emission. In the example shown, the fluorescence signal is amplified. In the figure, microtiter wells A and C contain no target protein-binding substance and therefore only a weak signal is detected. In contrast, a strongly increasing fluorescence signal is measured in recess B, since this recess contains a substance with the desired target-specific properties.
  • Figure 6 Secondary structures of reporter ribozyme construct IRK1 and ribozyme substrate SK1.
  • Rev binding sequence that of D.P. Bartel et al. isolated wt RBE motif selected [Cell 67 (1991), 529-536].
  • Figure 7 Secondary structures of the library (A) and the isolated Rev-binding sequence (Seq 5) (B) [L. Giver et al., 1993, NAR 23, 5509-5516].
  • FIG. 8a shows an allosteric hammerhead ribozyme which comprises an aptamer part which binds specifically to Rev. If Rev binds to the aptamer part of the ribozyme, the catalytic activity of the ribozyme is reduced.
  • a suitable FRET substrate in the present case consisting of the two components FAM and TAMRA, is used, energy is transferred between FAM and TAMRA when irradiated at a wavelength of 488 nm without fluorescence emission.
  • This fluorescence emission represents the signal which indicates that the respective compound, in the present example a small molecule, which is also included in this example is referred to as a candidate compound, for a specific interaction with leads to the target molecule and is therefore a lead substance for the target molecule in question.
  • the Rev protein with 1 ⁇ M can be used.
  • FIG. 8a shows the polynucleotide according to the invention, which is an allosteric aptazyme, in a method according to the invention for identifying a compound which interacts with a target molecule.
  • the ribozyme is in a catalytically active form.
  • the FRET-Susbtrate with FAM and TAMRA bound to the ribozyme and in particular the catalytically active domain is cleaved under the influence of the catalytic activity, the fluorescence quenching being abolished and fluorescence emission occurring at 520 nm when light of wavelength 480 nm is irradiated , If one or more compounds to be identified are added to such a reaction batch and the one or one of the compounds interacts with the target molecule, in the present case with the Rev protein, Rev will dissociate from the aptamer part of the polynucleotide according to the invention.
  • a pattern of base pairings which is different from the state in which the Rev protein was bound to the aptamer part is formed, thereby reducing or suppressing the catalytic activity of the ribozyme.
  • the substrate can no longer be cleaved and there is no longer any fluorescence emission, preferably due to the change in the base pairing pattern the substrate can no longer bind to the region of the catalytic activity.
  • FIG. 9 shows the sequences of the ribozymes according to the invention used for the screening assays described in Example 3, which are complexed with a 13mer substrate.
  • the aptamer-inhibited ribozyme AIR consists of a Rev aptamer which is linked via a penta-A linker to the 5 'end of the hammerhead ribozyme (HHR), which is a recently discovered hammerhead ribozyme (1) and (2).
  • the aptamer domain forms a helix with the substrate binding site of the ribozyme domain.
  • the aptamer part is not folded, but forms a helix with strain I of the ribozyme and thus prevents binding of the substrate.
  • the Rev response ribozyme (RRR) shown in (3) of FIG. 1 contains an HIV genomic Rev binding element (RBE) in strain II. RRR is active in the absence of Rev and is inhibited in its presence.
  • the three structures shown in FIG. 1 were folded according to the mfold server algorithm by M. Zucker and show the structures with minimal energy, namely 27.1 and 30.6 kcal / mol for AIR and 25.4 kcal / mol for RRR , 3 ' ends and ribozymes and 5 ' ends of the substrate were connected by a GAAA tetraloop for folding.
  • Figure 10 shows the initial reaction rates, expressed as measured fluorescence per time, for the first five minutes with the fluorescence removed from the negative control. Shown are reactions with only ribozyme and FRET substrate, Rev reactions which contain Rev and 1 ⁇ M Rev peptide in the case of HHR and RRR, 250 nM Rev in the case of AIR and reactions with Rev + compound 21 (Comp. 21) Compound 21, coumermycin A1 at 100 ⁇ M.
  • Rev leads to inhibition of the RRR ribozyme and activation of AIR.
  • the effect of Rev is completely reversed by 100 ⁇ M coumermycin in the case of RRR and AIR. Wild type HHR is not significantly affected by Rev and Compound 21 and serves as an internal control.
  • Figure 11 shows the concentration dependence of the effects of the identified compounds on Rev-binding screening ribozymes, RRR and AIR.
  • the initial reaction rates were determined by the initial increase in fluorescence divided by the rate of the ribozyme reaction in the active state where compounds were missing.
  • 11a shows reactions which contain 1 ⁇ M Rev peptide and various concentrations of coumermycin, novobiocin, nosiheptide and patulin.
  • Figure 11b shows the AIR reactions containing 250 nM Rev peptide and various concentrations of coumermycin, novobiocin and patulin.
  • FIG. 12 shows filter binding studies, the filter binding being carried out with 100 nM Rev protein, traces of 5 ' - 32 P-labeled reporter ribozymes, 200 nM FRET substrate and various concentrations of compounds in the test buffer at room temperature. The values shown are divided by a control reaction which contains no compound.
  • Figure 12a particularly shows binding studies with RRR, whereas Figure 12b shows binding studies with AIR.
  • FIG. 13 shows examples of the binding of identified antibiotics to Rev in the context of surface plasmon resonance studies.
  • Figure 13a shows the binding of coumermycin to surfaces derivatized with Rev peptide and Rev protein. No binding to RRR-derivatized surfaces is detectable.
  • Coumermycin only binds to Rev, not to ribozymes.
  • 13b shows the interaction of novobiocin, rosamicin, griseofulvin, streptolydigin and patulin with the Rev protein-derivatized surface. Only the antibiotics novobiocin and patulin that were identified in the screening show binding to Rev.
  • FIG. 14 shows the cleavage activity, which was measured by fluorescence per minute in the initial phase (5 min). Negative control reactions without ribozyme were subtracted.
  • Figure 14a shows the initial cleavage activity as it depends on Rev peptide for RRR and AIR. HHR is also slightly inhibited at more than 1 ⁇ M Rev peptide.
  • 14b shows the initial cleavage activity as a function of Rev protein for RRR and AIR. HHR is also inhibited above 2 ⁇ M Rev protein. Inhibition of AIR at high concentrations by Rev protein. 14a for the Rev peptide is not observed.
  • Figure 15 shows the screening results, where the ratios RRR / HHR and AIR / HHR were generated by subtracting the fluorescence of the negative control reactions without ribozyme from the initial fluorescence and dividing by the values obtained by active states of the ribozymes. Then RRR / AIR values were divided by HHR values to eliminate general effects on ribozyme function.
  • the batches contained 10 nM ribozyme, 200 nM substrate, 1 ⁇ M (Screen 1) or 250 nM (Screen 2) Rev peptide and 100 ⁇ M antibiotics.
  • Figure 16 shows Tables 1a and 1b.
  • Table 1a shows a comparison of the different values determined for selected antibiotics.
  • the "Screening results" column Identification by means of positive (RRR) and negative (AIR) measurement results, the values shown representing initial, corrected fluorescence per time in relation to the active state of the ribozymes and divided by relative HHR reactions.
  • Novobiocin was not included in the library
  • K CO mp Antibiotic concentrations for half maximal recovery (RRR) or inhibition (AIR) of the cleavage activity obtained by adjusting the data shown in Fig. 11.
  • Kfjiter Antibiotic concentration for half maximal resolution of the ribozyme / Rev- Protein interaction obtained by fitting the data shown in Figure 12.
  • Table 1b Kp values determined by surface plasmon resonance for antibiotic binding to the Rev protein.
  • FIG. 17 shows the method shown in FIG. 8, the secondary structures being shown.
  • the reporter system shown in Fig. 17 (A) corresponds to that shown in Fig. 8 (A) and the reporter system shown in Fig. 17 (B) corresponds to that shown in Fig. 8 (B).
  • Example 1 Screening of binders for the A-site subdomain of 16S RNA
  • a library of 500 non-characterized, low molecular weight substance mixtures (molecular weight less than 3000 g / mol) in microtiter plate format was tested with regard to their binding properties against a target ribozyme construct TRK1.
  • the substance mixtures were obtained by filtration of bacterial extracts (Actinomycetes strains).
  • the TRK1-RNA and the substrate SK1 were produced by automated solid phase synthesis (sequences see Figure 1).
  • the principle of operation of the assay is shown in FIG.
  • An inhibitory / activatory effect on the cleavage activity of the ribozyme domain was determined by a reduced / increased fluorescence signal.
  • a representative result of the screening experiment is shown in Table 1.
  • a schematic representation of the assay is shown in FIG. 3.
  • control batches without a target ribozyme construct TRK1 were measured on the same microtiter plate in addition to the respective measurement.
  • By subtracting the fluorescence signals measured in the control batches it was possible to largely eliminate ribozyme or target-unspecific influences of the respective substance on the FRET substrate (e.g. RNA aggregation, quenching effects or RNase degradation).
  • the measurement errors in determining the respective reaction speeds could be significantly reduced by correcting these non-specific influences.
  • Incubation with substance # 332, for example led to a significant reduction in the fluorescence signal in the control batch (FIG. 4b).
  • an increase in fluorescence was measured not only in the HHR1 reaction, but also in the control batch (FIG. 4c).
  • the ribozyme domain (HHR1) of the TRK1 was investigated separately, that is to say without a link sequence and a 16S RNA domain, in experiments carried out identically.
  • the missing sequence was replaced by the 5'-CCGGAUUGCCGG-3 'hairpin structure.
  • Substance # 122 was shown to inhibit the ribozyme HHR1, whereas substance # 387 had no measurable effect on the ribozyme.
  • substance # 122 was confirmed as a high-affinity and specific binder for the A-site subdomain.
  • Table 1 Representative results of the screening experiment (substances No97 - No192). The relative activity of the reporter ribozyme activity is listed in the presence of 100 ⁇ M of the respective substance. The values were obtained by regression analysis and subsequent normalization, the initial speed for the non-inhibited reaction being set to "1". Substances with a clear inhibitory influence are highlighted. All tests were carried out with substrate excess with 8 nM TRK1, 200 nM SK1 carried out in 0.5 x PBS at 32 ° C., 8 mM MgCl 2. The reactions were started by simultaneously adding MgCl 2 and substance.
  • Table 1 Representative results of the screening experiment (substances '97 -197)
  • RNA sequences For the screening of a substance that binds to the viral Rev protein, a library of 50 short RNA sequences was screened using the method according to the invention. The sequences are shown below.
  • the DNA matrices coding for the 50 RNA sequences were prepared by in vitro transcription of oligonucleotides synthesized by solid phase synthesis. After the transcription, the RNAs were separated by their length using denaturing polyacrylamide electrophoresis in denaturing urea / polyacrylamide gels, and the corresponding bands were visualized by fluorescence quenching. For this purpose, the gels were packed in transparent film, placed on DC aluminum foil (silica gel 60 F245, Merck) and irradiated with a hand lamp at 2 ⁇ 4 nm. Bands of the correct length were cut out, the gel pieces were broken up and covered with 300 mM sodium acetate (pH ⁇ , 2).
  • the gel suspension was pressed by syringes stuffed with glass wool. After removal of the gel residues, the nucleic acids were precipitated and taken up in sterile H 2 0.
  • FIG. 7 shows a general formula for the sequences from the RNA library which do not bind to the Rev protein, and the identified Rev-binding RNA sequence. The experiment was carried out as follows.
  • the sequences of the library and the IRK-1 reporter construct were denatured separately in reaction buffer (10 mM HEPES (pH 7.4), 100 mM NaCl) for 1.5 minutes at 95 ° C. and cooled again at room temperature for renaturation.
  • the IRK-1 and SK-1 were mixed and incubated for 5 min at room temperature. This incubation was then distributed evenly over 50 individual reactions. After the Rev protein had been added to the batches, the mixture was incubated for a further 15 min at room temperature before the 50 library RNA sequences were added to the ⁇ O batches. After a further 30 minutes, the cleavage reaction for the SK-2 substrate was started by adding magnesium.
  • the final concentrations of the final batches are composed as follows: 10 mM HEPES (pH 7.4), 100 mM NaCI, 10 mM MgCI 2) IRK-1 8 nM, SK-1 200 nM, RNA sequences from the library 2 ⁇ M, Rev protein 200 nM. All components were preheated to 37 ° C before mixing.
  • the evaluation was carried out as in Example 1 and identified the sequence in FIG. 7 as a Rev-binding sequence which could compete with the IRK-1 for binding to the protein.
  • the identification was made by measuring the significantly changed fluorescence compared to the approaches with other members of the library. This experiment was able to demonstrate that competitors of an RNA / protein interaction by the invention
  • the method can be isolated from combinatorial libraries easily and by means of fluorescence detection compatible with HTS methods.
  • Example 3 Construction of a high-throughput screening system for the identification of compounds interacting with the target protein Rev
  • Rev protein of HIV The interaction between the Rev protein of HIV and RRE facilitates the export of unspliced viral mRNA, which is important for viral replication and is therefore the subject of many approaches in the development of antiviral therapies [Dayton, A.I. & Zhang, M.J., Therapies directed against the Rev axis of HIV autoregulation, Adv Pharmacol 49, 199-228 (2000); Pollard, V.W. & Malim, M.H., The HIV-1 Rev protein, Annu Rev Microbiol 52, 491-532 (1998)].
  • two different hammerhead ribozymes were constructed, as shown in their secondary structure in FIG. 9 and the basic mode of operation in FIG. 8.
  • aptamer-inhibited ribozyme AIR
  • Rev aptamer-inhibited ribozyme AIR
  • Rev aptamer RNA aptamers selected to bind human immunodeficiency virus type 1 Rev in vitro are Rev responsive in vivo, J Virol 70, 179-87 (1996)] to the 5 'end of the ribozyme using a penta-A linker constructed.
  • the second ribozyme is inhibited by Rev.
  • Ribozyme (10 nM) and FRET-labeled substrate (200 nM) were previously incubated at room temperature for 5 minutes in test buffer, followed by optional addition of Rev and / or antibiotic. The reactions were then incubated for 5 min at 32 ° C and started by adding MgCl 2 to a final concentration of 8 mM through the dispenser function of the fluorescence meter. Negative controls with reactions that lacked the ribozyme were always included and were subtracted after the measurements.
  • Reactions (20 ⁇ l) containing 100 nM Rev protein, traces of ⁇ '- 32 P-labeled RRR or AIR, 200 nM FRET substrate and various concentrations of antibiotics in test buffer were washed through previously washed 0.4 ⁇ ⁇ m nitrocellulose membranes followed by washing with 3 ml of test buffer. The order of addition and incubation times were carried out similarly to the ribozyme reactions.
  • Rev protein and peptide surfaces were prepared by injecting 100 ⁇ M solutions in 60 mM NaOAc, pH 5.7 on EDC / NHS activated CM ⁇ chips, giving 1800 RU of immobilized peptide and ⁇ OOO RU protein.
  • the RRR-RNA surface was generated by injecting biotinylated RRR (75 nM) in Tris pH 7, ⁇ , 0, ⁇ M NaCI onto a streptavidin-derivatized chip and gave 1600 RU immobilized RRR. 4 ⁇
  • Both new Apatazyme AIR and RRR are regulated by means of the RNA domain for the Rev protein both by the complete Rev protein and by the Rev peptide comprising amino acids 34 to ⁇ O of the Rev protein.
  • the Kp values determined by means of surface plasmon resonance methods were RR n / Rev protein 1, 4 nM, RRR / Rev peptide 9.3 nM, AIR / Rev protein 1, 3 nM and AIR Rev peptide 9.0 nM.
  • AIR is also inhibited at Rev-Petid concentrations of more than 800 nM (cf. FIG. 14 a). This effect can be explained by an unspecific inhibition of the catalytic ribozyme function by the strongly positively charged peptide.
  • Even HHR is inhibited at Rev concentrations greater than: 1 ⁇ M. As shown in Figure 11, the initial activity is 93% at a concentration of 1 ⁇ M, although the cleavage is inhibited after a few minutes.
  • AIR itself remains activated up to concentrations of more than ⁇ ⁇ M, whereas RRR is inhibited above 1 ⁇ M and HHR above 2 ⁇ M Rev protein (cf. FIG. 14b).
  • Each 96-well plate contained the following standard duplicate reactions: ribozymes (HHR and reporter construct) alone, ribozyme reactions containing Rev peptide (1 ⁇ M in the case of RRR screening, 0.2 ⁇ ⁇ M in the case of AIR screening), two negative controls without ribozymes, which firstly only contained substrate and secondly contained Rev and substrate. Any reaction that contained antibiotics also contained Rev peptide. For each antibiotic, three different reactions, HHR, reporter construct (RRR or AIR), negative controls containing only antibiotics, and Rev and substrate were carried out. The antibiotic concentrations were 100 ⁇ M in each case.
  • FIG. 1 ⁇ A total of two approaches (English screens) were carried out, the result of which is shown in FIG. 1 ⁇ . Firstly, RRR, inhibited by 1 ⁇ M Rev peptide, was used, in which case active compounds were identified by restoring the catalytic activity of the ribozyme-mediated cleavage. On the other hand, the ribozyme AIR was used, whereby compounds, which are able to prevent the interaction between REV and the Rev aptamer, thereby identified that the Rev reporter construct, which is activated by 2 ⁇ 0 nM Rev peptide, is inhibited. The most effective hammerhead ribozyme inhibitors, antibiotics # 8, # 31, # 91 and # 92, could not be tested because they completely inhibited the response.
  • inactive compounds produce readings of approximately “zero” as a result of the inhibition by Rev.
  • Active compounds show a re-activation of the reporter construct RRR; complete re-activation results in a reading of 1.
  • Three compounds were identified which led to a significant one Activation (higher than 0.7 ⁇ ) resulted, namely coumermycin A1 (# 21), nosiheptide (# ⁇ 8) and patulin (# 63), as also shown in Table 1a.
  • inactive compounds In the AIR approach, inactive compounds have a value of approximately 1 because they leave the reporter construct in its active, Rev-binding state. Active compounds are identified by the fact that they give small readings close to zero (complete inhibition). Remarkably, the same compounds (# 21, # ⁇ 8 and # 63) were identified in a screening of the 96 antibiotic compound library and thus confirmed as Rev inhibitors.
  • Fig. 11 shows the results of examining the concentration dependency of the effect of breaking the interaction between Rev and the ribozyme.
  • Novobiocin a natural analogue of Coumermycin A, was included in all further studies. The results of the study of the relative initial speeds are summarized in Table 1a.
  • Rev protein was used instead of Rev peptide because the peptide is too small to be retained on the filter.
  • ⁇ O - 60% of the ⁇ '- 32 P-labeled reporter ribozymes RRR and AIR were retained on the filter in the presence of 100 nM Rev protein.
  • Adding identified antibiotics again confirmed the ability of the selected antibiotics to disrupt the interaction between RNA and the protein (see FIG. 12).
  • the results observed by fluorescence measurements with Rev peptide are confirmed by the results of the filter binding studies with Rev protein.
  • nucleic acid-binding protein from HIV-1 was tested with the target protein reverse transcriptase in order to check the specificity of the Rev-binding antibiotics identified. No significant effects were observed at concentrations of 100 ⁇ M antibiotics in a standard transcriptase assay (while ddCTP showed complete inhibition at 2. ⁇ ⁇ M).

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Abstract

L'invention concerne un procédé pour identifier des liaisons (structures initiales) qui (a) lient spécifiquement un motif cible ARN souhaité et inhibent ou éliminent ainsi sa fonction, ou bien qui (b) supplantent une liaison associée à un motif cible ARN souhaité et inhibent ou éliminent ainsi sa fonction. Le procédé selon l'invention est basé sur l'addition d'un ligand (= liaison à identifier) à un motif cible ARN couplé à un ribozyme modifié, de sorte que ce ribozyme est transformé en une conformation active ou inactive, ce qui provoque la division d'un substrat de ribozyme émetteur de signaux. Les liaisons ainsi identifiées, grâce auxquelles la fonction cellulaire des motifs cibles ARN peut être modifiée, permettent de fabriquer des médicaments spécifiques. La présente invention porte également sur un polynucléotide comprenant un ribozyme en « tête de marteau » et un aptamère pour une molécule cible, le modèle d'appariement de bases du polynucléotide, lorsqu'il y a liaison de la molécule cible à l'aptamère, se différenciant du modèle du polynucléotide en l'absence de la molécule cible sur l'aptamère.
EP01985820A 2000-11-22 2001-11-21 Procede pour identifier des liaisons ou des structures initiales contre des motifs cibles arn et des interactions arn/proteine Withdrawn EP1356105A2 (fr)

Applications Claiming Priority (5)

Application Number Priority Date Filing Date Title
DE10057853 2000-11-22
DE2000157853 DE10057853A1 (de) 2000-11-22 2000-11-22 Verfahren zur Identifizierung von Verbindungen oder Leitstrukturen gegen RNA-Target-Motive und RNA/Protein Wechselwirkungen
DE2001144355 DE10144355A1 (de) 2001-09-10 2001-09-10 Verfahren zur Identifizierung von Verbindungen oder Leitstrukturen gegen RNA-Target-Motive und RNA/Protein Wechselwirkungen
DE10144355 2001-09-10
PCT/EP2001/013568 WO2002042491A2 (fr) 2000-11-22 2001-11-21 Procede pour identifier des liaisons ou des structures initiales contre des motifs cibles arn et des interactions arn/proteine

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AU2002342672A1 (en) * 2001-09-11 2003-03-24 Nascacell Gmbh Method for screening for inhibitors of protein/protein interaction and corresponding ribozymes
WO2008127382A2 (fr) * 2006-10-19 2008-10-23 Yale University Dessin informatique de ribozymes
PL212696B1 (pl) * 2008-02-14 2012-11-30 Inst Chemii Bioorg Pan Inhibitor rybonukleazy Dicer

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EP0707638A4 (fr) * 1992-12-04 1998-05-20 Innovir Lab Inc Acide nucleique regulable a usage therapeutique et procedes d'utilisation associes
EP0948632A1 (fr) * 1996-10-31 1999-10-13 Smithkline Beecham Corporation Procedes de caracterisation et de selection de motifs cibles d'arn liant des composes pharmaceutiques

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