WO2002003919A2 - Cibles structurelles dans la sequence ires du virus de l'hepatite c - Google Patents

Cibles structurelles dans la sequence ires du virus de l'hepatite c Download PDF

Info

Publication number
WO2002003919A2
WO2002003919A2 PCT/US2001/021871 US0121871W WO0203919A2 WO 2002003919 A2 WO2002003919 A2 WO 2002003919A2 US 0121871 W US0121871 W US 0121871W WO 0203919 A2 WO0203919 A2 WO 0203919A2
Authority
WO
WIPO (PCT)
Prior art keywords
ires
loop
data
computer
domain
Prior art date
Application number
PCT/US2001/021871
Other languages
English (en)
Other versions
WO2002003919A3 (fr
WO2002003919A9 (fr
Inventor
Joseph D. Puglisi
Original Assignee
The Board Of Trustees Of The Leland Stanford Junior University
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by The Board Of Trustees Of The Leland Stanford Junior University filed Critical The Board Of Trustees Of The Leland Stanford Junior University
Priority to US10/332,626 priority Critical patent/US20040073380A1/en
Priority to AU2001273365A priority patent/AU2001273365A1/en
Publication of WO2002003919A2 publication Critical patent/WO2002003919A2/fr
Publication of WO2002003919A3 publication Critical patent/WO2002003919A3/fr
Publication of WO2002003919A9 publication Critical patent/WO2002003919A9/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C12Q1/701—Specific hybridization probes
    • C12Q1/706—Specific hybridization probes for hepatitis
    • C12Q1/707—Specific hybridization probes for hepatitis non-A, non-B Hepatitis, excluding hepatitis D

Definitions

  • Hepatitis C virus is spread primarily through contact with infected blood and can cause cirrhosis, irreversible and potentially fatal liver scarring, liver cancer, or liver failure. It can lie dormant for 10 years or more before symptoms appear. Some patients will have no symptoms of liver damage, and their liver enzymes will stay at normal levels. Other patients have severe hepatitis C, with detectable HCV in their blood, liver enzymes elevated as much as 20 times more than normal, and a prognosis of ultimately developing cirrhosis and end-stage liver disease. The disease is responsible for between 8,000, and 10,000 deaths yearly, and is the major reason for liver transplants in the United States, accounting for 1 ,000 of the procedures annually. Chronic hepatitis C varies widely in its severity and outcome.
  • HCV hepatitis C infection
  • the hepatitis C genome has an interesting feature, in that it possesses an internal ribosome entry site.
  • Normal translation initiation in eukaryotes occurs by recognition of the 5' cap structure of the mRNA by elF4E followed by assembly of other initiation factors and the 40S ribosomal subunit, and subsequent scanning of the 5' UTR to the first AUG initiation codon.
  • certain viral and other RNAs have been found to initiate translation in the absence of the 5' cap, through an internal ribosome entry site (IRES).
  • RNA RNA molecules
  • the hepatitis C virus genome is included in this group (see Tsukiyama-Kohara et al. (1992) J. Virol. 66:1476-83; and Wang ef al. (1993) J. Virol. 67:3338-44).
  • IRES-mediated initiation eliminates the requirement for the 5' cap structure and scanning.
  • the 40S subunit is recruited directly to the vicinity of the start codon by interaction with the IRES element; and only a subset of the total translation initiation factors is required for this process.
  • IRES-mediated initiation in HCV and related pestiviruses is reminiscent of prokaryotic translational initiation, in which the Shine-Dalgarno interaction recruits 30S subunits directly to the start codon, and only 3 initiation factors are required.
  • the IRES structure is unique to the virus, and so IRES-mediated translation is a potential drug target that can be exploited for inhibition of virus growth and replication.
  • This invention describes the identification and three-dimensional structures of RNA domains that are required for IRES activity. The structures can be used for rational design of ligands to target IRES activity.
  • the HCV IRES element is a complex RNA secondary structure consisting of nucleotides 44 to 354 in the 5'UTR (Honda et al. (1996) Virology 222, 31-42). Biochemical experiments have demonstrated the roles of particular subdomains in IRES function. In particular, mutagenesis and functional data support a pseudoknot structure near the AUG start codon (Wang ef al. (1995) RNA 1, 526-37). The sites of interaction with the 40S subunit have been mapped by toe-printing the subdomains including and adjacent to the pseudoknot structure (Pestova et al. (1998) Genes Dev. 12, 67-83).
  • GBV-B GB virus B
  • HCV hepatitis C virus
  • IRES internal ribosome entry site
  • HCV IRES hepatitis C virus IRES
  • the methods of the invention entail structural modeling, and the identification and design of molecules having a particular structure.
  • the methods rely on the use of precise structural information derived from NMR studies of the HCV IRES. This structural data permits the identification of atoms that are important for 40S ribosomal subunit binding.
  • Other molecules that include atoms having a similar three dimensional arrangement similar to the interaction surface between the 40S subunit and the IRES are likely to be capable of blocking this interaction.
  • Figure 1A shows the sequence and secondary structure of HCV IRES RNA (nucleotides 1-383 of HCV genotype 1b; SEQ ID NO. ). Domains are numbered according to Brown ef al. (1992) N.A.R. 20:5041-5045. Nucleotides protected from kethoxal modification upon 40S subunit binding are indicated. Nucleotides that show increases in DMS modification upon 40S subunit binding (O) are indicated.
  • Figure 1(b) is an autoradiograph of kethoxal and DMS probing of HCV IRES RNA domains Illd and llle in the absence (-), or presence (+) of 40S subunits.
  • the K lane is a primer extension reaction using the unmodified HCV IRES RNA.
  • the kethoxal (ket) and DMS probing and primer extension reactions are performed as described under methods.
  • the lanes marked U, G, C, and A are dideoxy sequencing reactions.
  • Figure 1 (c) Sequence and secondary structure of the HCV IRES domain llle (SEQ ID NO:2) and Illd (SEQ ID NO:3) RNA oligonucleotides used for the NMR structural studies. Numbering according to Fig. 1a. Nucleotides, that were changed to improve transcription efficiency, are outlined.
  • Figure 2(a) shows a stereo view from the major groove of the heavy-atom superposition of final 20 structures of HCV IRES domain llle. Bases are colored in blue and ribose-phosphate backbone in gray.
  • Figure 2(b) shows a single representative structures of the GNRA (SEQ ID NO:3) (Heus and Pardi (1991 ) Science 253:191-194) and the GAUA (SEQ ID NO:4) tetraloop of HCV IRES domain llle. Base nitrogens are in blue and base oxygens in red. Phosphorus atoms and phosphate oxygens are shown explicitly in yellow and red, respectively.
  • Figure 3(a) is a stereo view of the heavy-atom superposition of final 25 structures of HCV IRES domain Illd. The color scheme is the same as in Fig. 2a.
  • Figure 3(b) is a single representative structure of the HCV IRES domain Illd, with both S turns highlighted. Ribose O4' atoms are shown in red, the inverted riboses in blue and the phosphate backbone in yellow.
  • Figure 3(c) is a major groove view of the heavy-atom superposition of hairpin loop nucleotides G263-C270 of the final 25 structures of the HCV IRES domain Illd and a single representative structure. The color scheme is the same as in Fig. 2a and 2b. Phosphorus atoms are shown in yellow.
  • Figure 3(d) is a minor groove view of the heavy-atom superposition of loop E nucleotides C255-G261 and C272-G277 of the final 25 structures of the HCV IRES domain Illd and a single representative structure omitting the flanking G-C base pairs.
  • the color scheme is the same as in Fig. 3c.
  • Figure 3(e) depicts the base pairing schemes found within the loop E motif of HCV IRES domain Illd.
  • the color scheme is the same as in Fig. 2a.
  • Hydrogen bonds shown by dashed lines are observed in all 25 final NMR structures.
  • Tables 4a, b and c contain the proton and 15N chemical shifts for HCV IRES domains.
  • the structure information may be provided in a computer readable form, e.g. as a database of atomic coordinates, or as a three-dimensional model.
  • the structures are useful, for example, in modeling interactions of the IRES with its binding partner, the 40S ribosome subunit.
  • the structures are also used to identify non-ribosomal molecules that bind to the IRES element, and block the interaction with the 40S subunit.
  • HCV IRES STRUCTURE The coordinates for the HCV IRES domains are can be obtained from Lukavsky ef al. (2000) Nat Struct Biol 7(12):1105-10. These coordinates can be used in the design of structural models and screening methods according to the methods of the invention.
  • RNA stem loops domains Illd and llle, are involved in IRES-40S subunit interaction, and are targets for blocking agents.
  • the domain llle hairpin loop adopts a novel tetraloop fold.
  • the hairpin is closed by a U294-G299 wobble pair, followed by a sheared G295-A298 base pair.
  • the loop sequence (-GAUA-) does not conform to the standard GNRA motif, and adopts a different structure, wherein the bases of A296 and U297 point towards the major groove, and are not involved in RNA backbone contacts that would stabilize the loop fold; the two central nucleotides of the tetraloop stack on the 5'guanosine of the sheared G-A pair.
  • This fold creates an array of three major groove exposed Watson-Crick faces (G295, A296, and U297). This loop was found to be a point of direct contact with the 40S subunit.
  • the domain Illd RNA forms a helical stem with non-canonical pairings, followed by a hexanucleotide loop region.
  • the -UUGGGU- hairpin loop is more disordered than the other regions of the RNA.
  • the central internal loop which is highly conserved among HCV isolates, adopts the well-characterized loop E fold.
  • Four consecutive non-canonical base pairs are formed - a sheared G256-A276 pair, a parallel A257-A275 pair, a reverse Hoogsteen U259-A274 pair and another sheared A260-G273 base pair.
  • the arrangement of the base pairs within the loop E motif creates a continuous stack of four adenines (A260 and A274 to A276) with their Watson-Crick faces exposed to the minor groove.
  • the internal loop is asymmetric, with the bulged G258 positioned in the major groove, where it forms a base triple with the U259-A274 reverse Hoogsteen pair.
  • the phosphodiester backbone undergoes a local reversion of direction at A257 and G258, such that a parallel hydrogen bonding arrangement between A257 and A275 can form.
  • the inversion in backbone direction leads to a characteristic S turn in the backbone between G256 to A259.
  • the unusual backbone geometry within the loop E motif results from non A-form values for torsion angles ⁇ for G258 and A274 (gauche + ), ⁇ for A274 (trans), and ⁇ for A257 (gauche + ) to allow for the triple formation and backbone inversion.
  • the sequence of the -UUGGGU-hairpin loop of domain Illd is absolutely conserved among all HCV isolates.
  • the hairpin loop is separated from the loop E motif by a short helical stem consisting of a G-U wobble pair flanked by two G-C Watson-Crick base pairs, of which one closes the hairpin loop.
  • U264 stacks on top of the loop closing G-C base pair, whereas U269 on the 3' side is bulged into solution and disordered in the ensemble of NMR structures.
  • a set of structure coordinates for a structure is a relative set of points that define a shape in three dimensions.
  • a set of structures was generated that are consistent with the NMR spectral analyses.
  • This range of structures (or ensemble) provided herein represents the error in measurement, and the real disorder in the actual structure due to the dynamic nature of the molecules. It is possible that an entirely different set of coordinates could define a similar or identical shape.
  • slight variations caused by acceptable errors in the individual coordinates will have little, if any effect on overall shape. In terms of binding grooves, these acceptable variations would not be expected to alter the nature of ligands that could associate with those structures.
  • the variations may be generated because of mathematical manipulations of the structure coordinates.
  • modifications in the NMR structure due to mutations, additions and deletions of nucleotides in the HCV RNA could also account for variations in structure coordinates.
  • the ensemble of structures represents an acceptable standard of error for the coordinates. If variations in a related structure are within an acceptable standard error as compared to the original coordinates, the resulting three-dimensional shape is considered to be the same.
  • a ligand that bound to the HCV IRES may also be expected to bind to another IRES element whose structure coordinates defined a shape that falls within the acceptable error.
  • IRES structure models and databases of structure information are provided.
  • the structure model may be implemented in hardware or software, or a combination of both.
  • a machine-readable storage medium comprising a data storage material encoded with machine readable data which, when using a machine programmed with instructions for using said data, is capable of displaying a graphical three-dimensional representation of any of the structures of this invention that have been described above.
  • the computer-readable storage medium is capable of displaying a graphical three- dimensional representation of the HCV IRES stem loops in one or both of domains Illd and llle.
  • data providing structural coordinates, alone or in combination with software capable of displaying the resulting three dimensional structure of the HCV IRES subdomains described above, portions thereof, and their structurally similar homologues, is stored in a machine-readable storage medium.
  • Such data may be used for a variety of purposes, such as drug discovery, analysis of interactions between cellular components during translation, modeling of vaccines, and the like.
  • the invention is implemented in computer programs executing on programmable computers, comprising a processor, a data storage system (including volatile and non-volatile memory and/or storage elements), at least one input device, and at least one output device.
  • Program code is applied to input data to perform the functions described above and generate output information.
  • the output information is applied to one or more output devices, in known fashion.
  • the computer may be, for example, a personal computer, microcomputer, or workstation of conventional design.
  • Each program is preferably implemented in a high level procedural or object oriented programming language to communicate with a computer system.
  • the programs can be implemented in assembly or machine language, if desired. In any case, the language may be a compiled or interpreted language.
  • Each such computer program is preferably stored on a storage media or device (e.g., ROM or magnetic diskette) readable by a general or special purpose programmable computer, for configuring and operating the computer when the storage media or device is read by the computer to perform the procedures described herein.
  • the system may also be considered to be implemented as a computer-readable storage medium, configured with a computer program, where the storage medium so configured causes a computer to operate in a specific and predefined manner to perform the functions described herein.
  • HCV BINDING PARTNERS AND MIMETICS The structure of the two HCV IRES stem loops, domains Illd and llle, are useful in the design of agents that block the interaction between the viral mRNA and
  • Agents of interest may comprise mimetics of the HCV IRES structure, which then compete for binding of the 40S subunit.
  • the agents of interest may be an HCV binding agent, for example a structure that directly binds to the HCV IRES by having a physical shape that provides the appropriate contacts and space filling in the grooves of one or both of the domains.
  • the structure encoded by the data may be computationally evaluated for its ability to associate with chemical entities. This provides insight into IRES element's ability to associate with chemical entities. Chemical entities that are capable of associating with these domains may inhibit translation from HCV mRNA. Such chemical entities are potential drug candidates.
  • the structure encoded by the data may be displayed in a graphical format. This allows visual inspection of the structure, as well as visual inspection of the structure's association with chemical entities.
  • a invention for evaluating the ability of a chemical entity to associate with any of the molecules or molecular complexes set forth above.
  • This method comprises the steps of employing computational means to perform a fitting operation between the chemical entity and the interacting surface of the RNA surface; and analyzing the results of the fitting operation to quantify the association.
  • chemical entity refers to chemical compounds, complexes of at least two chemical compounds, and fragments of such compounds or complexes.
  • Molecular design techniques are used to design and select chemical entities, including inhibitory compounds, capable of binding to HCV IRES, particularly domain Illd and/or lie.
  • Such chemical entities may interact directly with certain key features of the HCV IRES structure, including, without limitation, the novel tetraloop fold in domain llle, including the array of three major groove exposed Watson-Crick faces (G295, A296, and U297), which loop has been found to be a point of direct contact with the 40S subunit.
  • inhibitory compounds may interact with the S turn in the backbone between G256 to A259, and particularly in the structure comprising the loop E and hairpin loop backbone reversion, which places both S turns on the same side of the hairpin loop structure.
  • Such chemical entities and compounds may interact with either or both structures, in whole or in part.
  • the compound must be able to assume a conformation that allows it to associate or compete with the IRES element. Although certain portions of the compound will not directly participate in these associations, those portions of the may still influence the overall conformation of the molecule. This, in turn, may have a significant impact on potency.
  • conformational requirements include the overall three-dimensional structure and orientation of the chemical entity in relation to all or a portion of the binding pocket, or the spacing between functional groups of an entity comprising several interacting chemical moieties.
  • Computer-based methods of analysis fall into two broad classes: database methods and de novo design methods.
  • database methods the compound of interest is compared to all compounds present in a database of chemical structures and compounds whose structure is in some way similar to the compound of interest are identified.
  • the structures in the database are based on either experimental data, generated by NMR or x-ray crystallography, or modeled three-dimensional structures based on two-dimensional data.
  • de novo design methods models of compounds whose structure is in some way similar to the compound of interest are generated by a computer program using information derived from known structures, e.g. data generated by x-ray crystallography and/or theoretical rules.
  • Such design methods can build a compound having a desired structure in either an atom-by- atom manner or by assembling stored small molecular fragments. Selected fragments or chemical entities may then be positioned in a variety of orientations, or docked, within the interacting surface of the RNA. Docking may be accomplished using software such as Quanta (Molecular Simulations, San Diego, CA) and Sybyl, followed by energy minimization and molecular dynamics with standard molecular mechanics force fields, such as CHARMM and AMBER.
  • Specialized computer programs may also assist in the process of selecting fragments or chemical entities. These include: GRID (Goodford (1985) J. Med. Chem., 28, pp. 849-857; Oxford University, Oxford, UK; MCSS (Miranker et al. (1991) Proteins: Structure, Function and Genetics, 11 , pp. 29-34; Molecular Simulations, San Diego, CA); AUTODOCK (Goodsell ef al., (1990) Proteins: Structure, Function, and Genetics, 8, pp. 195-202; Scripps Research Institute, La Jolla, Calif.); and DOCK (Kuntz ef al. (1982) J. Mol. Biol., 161:269-288; University of California, San Francisco, Calif.)
  • suitable chemical entities or fragments can be assembled into a single compound or complex. Assembly may be preceded by visual inspection of the relationship of the fragments to each other on the three- dimensional image displayed on a computer screen in relation to the structure coordinates.
  • Useful programs to aid one of skill in the art in connecting the individual chemical entities or fragments include: CAVEAT (Bartlett et al. (1989) In Molecular Recognition in Chemical and Biological Problems", Special Pub., Royal Chem. So ⁇ , 78, pp. 182-196; University of California, Berkeley, Calif.); 3D Database systems such as MACCS-3D (MDL Information Systems, San Leandro, Calif); and HOOK (available from Molecular Simulations, San Diego, CA).
  • substitutions may then be made in some of its atoms or side groups in order to improve or modify its binding properties.
  • initial substitutions are conservative, i.e., the replacement group will have approximately the same size, shape, hydrophobicity and charge as the original group. It should, of course, be understood that components known in the art to alter conformation should be avoided.
  • substituted chemical compounds may then be analyzed for efficiency of fit by the same computer methods described above.
  • Another approach made possible and enabled by this invention is the computational screening of small molecule databases for chemical entities or compounds that can bind in whole, or in part, to the IRES element. In this screening, the quality of fit of such entities to the binding site may be judged either by shape complementarity or by estimated interaction energy.
  • BIOLOGICAL SCREENING The success of both database and de novo methods in identifying compounds with activities similar to the compound of interest depends on the identification of the functionally relevant portion of the compound of interest.
  • the functionally relevant portion may be referred to as a pharmacophore, i.e. an arrangement of structural features and functional groups important for biological activity.
  • Not all identified compounds having the desired pharmacophore will act as an inhibitor of HCV translation.
  • the actual activity can be finally determined only by measuring the activity of the compound in relevant biological assays.
  • the methods of the invention are extremely valuable because they can be used to greatly reduce the number of compounds which must be tested to identify an actual inhibitor.
  • the binding assay will further comprise a 40 S ribosome subunit or fragment derived therefrom, where binding of the ribosome subunit to the HCV IRES is quantitated.
  • biological assays may be performed. For example, one may test the effect of a candidate compound on protein translation, by monitoring the synthesis of HCV proteins in the presence or absence of the candidate agent. Agents may also be tested in a cellular setting, for example to monitor the production of viral particles in the absence or presence of the candidate agent.
  • EXPERIMENTAL The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the subject invention, and are not intended to limit the scope of what is regarded as the invention. Efforts have been made to ensure accuracy with respect to the numbers used (e.g. amounts, temperature, concentrations, etc.) but some experimental errors and deviations should be allowed for.
  • Fig. 1 b Upon binding of the folded IRES to 40S subunits, two regions of the IRES show strong changes in reactivity (Fig. 1 b). Guanosines in the hairpin loops of domains Illd and llle are strongly reactive to kethoxal in the unbound IRES, and are almost completely protected upon 40S binding. In contrast, three adenosines in the stem of domain Illd show increases in reactivity upon 40S subunit binding. These loops are required for IRES function in vivo and in vitro.
  • RNA oligonucleotide corresponding to nucleotides 291 to 302 was studied using homonuclear NMR methods. The spectra were readily assigned without isotopic labeling, and a total of 271 NOE and 88 dihedral torsion angle restraints were obtained.
  • domain Illd a 29 residue RNA oligonucleotide, corresponding to nucleotides 253 to 279, was studied by NMR spectroscopy.
  • a combination of homonuclear and heteronuclear 2D and 3D NMR methods yielded a total number of 705 NOE and 200 dihedral torsion angle restraints. Measurement of intra base pair 2 J N N couplings across hydrogen bonds was employed to establish base pairing schemes. For both hairpin loops, solely NMR-derived restraints were used for structure calculations.
  • the hairpin is closed by a U294-G299 wobble pair, followed by a sheared G295-A298 base pair (Fig. 2b).
  • Similar pairing interactions are observed in the GNRA tetraloops and in purine-rich hexaloops.
  • the loop sequence (-GAUA-), however, does not conform to the standard GNRA motif, and adopts a different structure.
  • the bases of A296 and U297 point towards the major groove, and are not involved in RNA backbone contacts that would stabilize the loop fold; the two central nucleotides of the tetraloop stack on the 5'guanosine of the sheared G-A pair.
  • This fold creates an array of three major groove exposed Watson-Crick faces (G295, A296, and U297).
  • the central purine and adenosine in a GNRA tetraloop point towards the minor groove, and are stacked on the 3'adenosine of the sheared G-A pair (Fig. 2b).
  • the sequence of the GAUA tetraloop is conserved among all HCV isolates, and among related pestiviridae IRES.
  • All dicistronic vectors contain an additional mutation of the HCV AUG start codon to CUG, which did not affect translational activity. All mRNAs displayed similar intracellular stabilities, as determined by Northern analysis.
  • the domain Illd RNA forms a helical stem with non-canonical pairings, followed by a hexanucleotide loop region.
  • the -UUGGGU- hairpin loop is more disordered than the other regions of the RNA; the heavy atom r.m.s.d. for U264 to U269 is 1.46A (Fig. 3c).
  • the central internal loop which is highly conserved among HCV isolates, adopts the well-characterized loop E fold, and is very well defined by the NMR data (r.m.s.d. of 0.28A, Fig. 3d).
  • the internal loop is asymmetric, with the bulged G258 positioned in the major groove, where it forms a base triple with the U259-A274 reverse Hoogsteen pair (Fig. 3e).
  • the phosphodiester backbone undergoes a local reversion of direction at A257 and G258, such that a parallel hydrogen bonding arrangement between A257 and A275 can form.
  • the inversion in backbone direction leads to a characteristic S turn in the backbone between G256 to A259 (Fig. 3b).
  • the unusual backbone geometry within the loop E motif results from non A-form values for torsion angles ⁇ for G258 and A274 (gauche + ), ⁇ for A274 (trans), and ⁇ for A257 (gauche + ) to allow for the triple formation and backbone inversion.
  • the structure also explains unusual 1 H, 13 C, and 31 P chemical shifts observed for several loop E resonances.
  • the loop E motif is common in RNAs, with different sequence families. All loop E motifs contain a sheared G-A pair and the adjacent U-A pair. In prokaryotic 5S ribosomal RNA, a loop E motif is observed with a symmetric internal loop: a G-G pair and sheared G-A pair. In eukaryotic 28S ribosomal RNA, the sarcin-ricin loop (SRL) contains a loop E motif that contains the A-A pair, bulged G, U-A pair and G- A pair; the r.m.s.d. between the crystal structure of SRL and the HCV IRES domain Illd loop E motif is 1.15 A.
  • the SRL has a flexible region adjacent to the loop E motif, whereas in domain Illd the loop E is bordered by a sheared G-A pair and Watson-Crick base pairs. Loop E motifs present rich hydrogen bonding potential in both the minor and major groove for both RNA-RNA and RNA-protein interactions.
  • the sequence of the -UUGGGU-hairpin loop of domain Illd is absolutely conserved among all HCV isolates.
  • the hairpin loop is separated from the loop E motif by a short helical stem consisting of a G-U wobble pair flanked by two G-C Watson-Crick base pairs, of which one closes the hairpin loop.
  • U264 stacks on top of the loop closing G-C base pair, whereas U269 on the 3' side is bulged into solution and disordered in the ensemble of NMR structures (Fig. 3c).
  • the six base pair spacing between the loop E and the hairpin loop backbone reversion places both S turns on the same side of the hairpin loop structure. This creates a unique backbone feature for the domain Illd motif.
  • the domain Illd hairpin loop clearly plays an important role in IRES-40S subunit interaction.
  • the Watson-Crick faces of G266, G267 and G268 were strongly protected from reaction with kethoxal in the IRES-40S subunit complex (Fig. 1 b).
  • the N7 positions of G266 and G267 are protected from methylation by dimethyl sulfate in the IRES-40S complex, whereas the N7 of G268, which is exposed on the major groove side of the Illd loop, is highly reactive in the complex.
  • the three guanosines in the loop are required for full IRES activity in internal initiation.
  • adenosine N1 positions of A274 to A276 in the minor groove of the loop E motif are highly accessible to modification by DMS in the IRES-40S subunit complex (Fig. 1 b). Therefore, protein- or RNA interactions with the loop E motif likely occur on the major groove side.
  • the results presented here demonstrate that two surface-accessible stem loops in the HCV IRES are involved in complex formation with 40S ribosomal subunits.
  • the novel structures of both domain Illd and llle are suggestive of their involvement in IRES function, and suggest experiments for a molecular level understanding of HCV IRES function.
  • the work presented here demonstrates the powerful ability of RNA NMR to provide local structural information to drive biochemical studies of a large RNA system.
  • RNA oligonucleotides were prepared by transcription from DNA templates by phage T7 RNA polymerase and purified using polyacrylamide gel electrophoresis (Puglisi ef al. (1995) Methods Enzymol. 261,
  • RNAs were electroeluted from the gel and subsequently dialyzed against final buffer (10mM Na phosphate, pH 6.4, 1mM d ⁇ 2 - EDTA, 4% D 2 O or 100% D 2 O).
  • NMR samples were prepared in a Shigemi NMR tube (sample volume 250 ⁇ L) at RNA concentrations of 1.0-2.5mM.
  • NMR Spectral analyses NMR data were acquired at either 15 or 25°C on Varian Inova 500 MHz and 800MHz NMR spectrometers equipped with triple resonance x,y,z-axis gradient probes. 1 H, 13 C, 15 N, and 31 P assignments were obtained using standard homonuclear and heteronuclear methods optimized for RNA structure determination (RnaPack, Varian User Library). In short, constant time HSQC, 3D HCCH-TOCSY, 3D HCCH-COSY, and 2D HCCH-RELAY experiments were used to assign sugar spin systems, while through-backbone assignments were made with HCP and HP-COSY experiments (Marino ef al. (1994) J. Am. Chem. Soc.
  • Base exchangeable protons were assigned by correlation to non- exchangeable base protons using heteroTOCSY methods.
  • Intranucleotide H1' to base proton correlations were obtained using a 2D MQ-HCN experiment (Marino etal. (1997) J. Am. Chem. Soc. 119, 7361-7366).
  • NOE distance restraints from non-exchangeable protons were obtained from 2D-NOESY experiments (100% D 2 O) with mixing times of 50, 150 and 250 ms.
  • Exchangeable proton NOEs were determined using SS-NOESY (Smallcombe (1993) J. Am. Chem. Soc.
  • Structure calculation Structures were calculated using a simulated annealing protocol within the X-PLOR 3.1 package (Br ⁇ nger, A . X-PLOR (Version 3.1) A System for X-ray Crystallography and NMR. Yale University Press, New Haven, CT (1993).
  • the protocol for structural calculations included two stages; simulated annealing of starting structures with random angles and restrained molecular dynamics (rMD) refinement.
  • rMD restrained molecular dynamics
  • NOE distance force constants were set to 50 kcal mol "1 A "2 and torsion angle force constants were varied from 5 to 50 kcal mol "1 rad "2 during calculations. No hydrogen bonding restraints other than experimentally measured ones were used in calculations.
  • a total of 100 starting structures were generated and subjected to a simulated annealing protocol. This consisted of 500 cycles of energy minimization, followed by rMD at 1000K with low values for interatomic repulsion, and subsequent rMD with increasing values for interatomic repulsion while cooling to 300K. A final minimization step with 1000 cycles was performed, which included a Lennard-Jones potential and no electrostatic terms.
  • the 100 structures were then subjected to a refinement procedure: 500 steps of restrained energy minimization; rMD at 1000K while increasing the torsion angle force constant; rMD while cooling to 300K and finally 1000 cycles of energy minimization, which included a Lennard-Jones potential, but no electrostatic terms.
  • HCV IRES RNA (nt 40-375) was generated by T7 RNA polymerase run-off transcription and purified by gel electrophoresis. Chemical modification with kethoxal and DMS was performed essentially as described in Moazed and Noller (1986) Cell 47, 985-94.
  • DNA plasmids were transfected into HeLa cells using the FuGENE 6 transfection reagent (Boehringer Mannheim). Transfected Cells were harvested 24 hours after transfection and luciferase activities were measured using the Dual Luciferase Reporter Assay System (Promega Biotech).
  • SA> refers to the final 20 simulated annealing structures, SA to the average structure obtained by taking the average coordinates of the 20 simulated annealing structures best-fitted to one another, + " the 20 final structures did not contain distance violations of > 0.25 A or dihedral violations of > 5°. Numbers in parentheses refer to number of restraints.
  • SA> refers to the final 25 simulated annealing structures, SA to the average structure obtained by taking the average coordinates of the 25 simulated annealing structures best-fitted to one another. + " the 25 final structures did not contain distance violations of > 0.25 A or dihedral violations of > 5°. Numbers in parentheses refer to number of restraints.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Communicable Diseases (AREA)
  • Immunology (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Molecular Biology (AREA)
  • Analytical Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Virology (AREA)
  • Biotechnology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Peptides Or Proteins (AREA)

Abstract

L'invention concerne des structures moléculaires, des représentations informatiques correspondantes et des procédés d'analyse se rapportant au site d'entrée du ribosome interne du virus de l'hépatite (VHC IRES). L'invention concerne également des éléments structurels comme le pli en tétraboucle du domaine IIIe, y compris la série de trois faces de Watson-Crick exposées à rainures majeures (G295, A296, et U297), cette boucle apparaissant comme un point de contact direct avec la sous-unité 40S; ou encore la structure à boucle E du domaine IIId et à réversion de squelette de boucle en épingle à cheveux, laquelle donne deux virages en S du même côté de la structure de boucle en épingle à cheveux.
PCT/US2001/021871 2000-07-10 2001-07-10 Cibles structurelles dans la sequence ires du virus de l'hepatite c WO2002003919A2 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
US10/332,626 US20040073380A1 (en) 2001-07-10 2001-07-10 Structural targets in hepatittis c virus ires element
AU2001273365A AU2001273365A1 (en) 2000-07-10 2001-07-10 Structural targets in hepatitis c virus ires element

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US21767300P 2000-07-10 2000-07-10
US60/217,673 2000-07-10

Publications (3)

Publication Number Publication Date
WO2002003919A2 true WO2002003919A2 (fr) 2002-01-17
WO2002003919A3 WO2002003919A3 (fr) 2002-04-18
WO2002003919A9 WO2002003919A9 (fr) 2003-04-03

Family

ID=22812031

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2001/021871 WO2002003919A2 (fr) 2000-07-10 2001-07-10 Cibles structurelles dans la sequence ires du virus de l'hepatite c

Country Status (2)

Country Link
AU (1) AU2001273365A1 (fr)
WO (1) WO2002003919A2 (fr)

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999017616A1 (fr) * 1997-10-07 1999-04-15 New England Medical Center Hospitals, Inc. Conception rationnelle de composes permettant de lutter contre les infections par papillomavirus, fondee sur leur structure
US5978740A (en) * 1995-08-09 1999-11-02 Vertex Pharmaceuticals Incorporated Molecules comprising a calcineurin-like binding pocket and encoded data storage medium capable of graphically displaying them
US6183121B1 (en) * 1997-08-14 2001-02-06 Vertex Pharmaceuticals Inc. Hepatitis C virus helicase crystals and coordinates that define helicase binding pockets

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5978740A (en) * 1995-08-09 1999-11-02 Vertex Pharmaceuticals Incorporated Molecules comprising a calcineurin-like binding pocket and encoded data storage medium capable of graphically displaying them
US6183121B1 (en) * 1997-08-14 2001-02-06 Vertex Pharmaceuticals Inc. Hepatitis C virus helicase crystals and coordinates that define helicase binding pockets
WO1999017616A1 (fr) * 1997-10-07 1999-04-15 New England Medical Center Hospitals, Inc. Conception rationnelle de composes permettant de lutter contre les infections par papillomavirus, fondee sur leur structure

Also Published As

Publication number Publication date
WO2002003919A3 (fr) 2002-04-18
AU2001273365A1 (en) 2002-01-21
WO2002003919A9 (fr) 2003-04-03

Similar Documents

Publication Publication Date Title
Berry et al. Crystal structure of the HCV IRES central domain reveals strategy for start-codon positioning
Batey et al. Tertiary motifs in RNA structure and folding
Lukavsky et al. Structures of two RNA domains essential for hepatitis C virus internal ribosome entry site function
Robertson et al. The structure of a rigorously conserved RNA element within the SARS virus genome
Hartmann et al. DNA structural forms
Lind et al. Structure-based computational database screening, in vitro assay, and NMR assessment of compounds that target TAR RNA
Du et al. Structure of TAR RNA complexed with a Tat-TAR interaction nanomolar inhibitor that was identified by computational screening
Boerneke et al. Physical and functional analysis of viral RNA genomes by SHAPE
US7438920B1 (en) Crystals of hepatitis C virus helicase or fragments thereof comprising a helicase binding pocket
Hu et al. The characteristic of codon usage pattern and its evolution of hepatitis C virus
Staple et al. Solution structure of the HIV‐1 frameshift inducing stem–loop RNA
JP2001507213A (ja) 核酸の診断検出
Stephenson et al. Three-dimensional RNA structure of the major HIV-1 packaging signal region
Gonzales et al. Synonymous–non-synonymous mutation rates between sequences containing ambiguous nucleotides (Syn-SCAN)
Rijnbrand et al. Mutational and structural analysis of stem-loop IIIC of the hepatitis C virus and GB virus B internal ribosome entry sites
Gallego et al. Rev binds specifically to a purine loop in the SL1 region of the HIV-1 leader RNA
Rybicka et al. Current molecular methods for the detection of hepatitis B virus quasispecies
Smith et al. A novel point mutation at position 156 of reverse transcriptase from feline immunodeficiency virus confers resistance to the combination of (−)-β-2′, 3′-dideoxy-3′-thiacytidine and 3′-azido-3′-deoxythymidine
Gosavi et al. Insights into the secondary and tertiary structure of the bovine viral diarrhea virus internal ribosome entry site
Drusano et al. Pharmacodynamics of abacavir in an in vitro hollow-fiber model system
US20040073380A1 (en) Structural targets in hepatittis c virus ires element
Parvez et al. Molecular analysis and modeling of hepatitis E virus helicase and identification of novel inhibitors by virtual screening
WO2002003919A2 (fr) Cibles structurelles dans la sequence ires du virus de l'hepatite c
US20020018988A1 (en) In silico screening
Moser et al. Quantifying mixed populations of drug-resistant human immunodeficiency virus type 1

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A2

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NO NZ PL PT RO RU SD SE SG SI SK SL TJ TM TR TT TZ UA UG US UZ VN YU ZA ZW

AL Designated countries for regional patents

Kind code of ref document: A2

Designated state(s): GH GM KE LS MW MZ SD SL SZ TZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE TR BF BJ CF CG CI CM GA GN GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
AK Designated states

Kind code of ref document: A3

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NO NZ PL PT RO RU SD SE SG SI SK SL TJ TM TR TT TZ UA UG US UZ VN YU ZA ZW

AL Designated countries for regional patents

Kind code of ref document: A3

Designated state(s): GH GM KE LS MW MZ SD SL SZ TZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE TR BF BJ CF CG CI CM GA GN GW ML MR NE SN TD TG

DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

COP Corrected version of pamphlet

Free format text: PAGES 1/11-11/11, DRAWINGS, REPLACED BY NEW PAGES 1/9-9/9; DUE TO LATE TRANSMITTAL BY THE RECEIVINGOFFICE

122 Ep: pct application non-entry in european phase
WWE Wipo information: entry into national phase

Ref document number: 10332626

Country of ref document: US

NENP Non-entry into the national phase in:

Ref country code: JP