WO2000075182A1 - Cristal de proteine du facteur de recyclage ribosomique (rrf) et son application sur la base de donnees de structure tridimensionnelles provenant du cristal - Google Patents

Cristal de proteine du facteur de recyclage ribosomique (rrf) et son application sur la base de donnees de structure tridimensionnelles provenant du cristal Download PDF

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WO2000075182A1
WO2000075182A1 PCT/JP2000/003639 JP0003639W WO0075182A1 WO 2000075182 A1 WO2000075182 A1 WO 2000075182A1 JP 0003639 W JP0003639 W JP 0003639W WO 0075182 A1 WO0075182 A1 WO 0075182A1
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rrf
protein
crystal
sat
amino acid
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PCT/JP2000/003639
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Japanese (ja)
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Akira Kaji
Anders Liljas
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Akira Kaji
Anders Liljas
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/37—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi

Definitions

  • the present invention relates to a crystal of a ribosome recycling factor (RRF).
  • RRF ribosome recycling factor
  • the present invention also relates to a three-dimensional structure of RRF protein obtained by X-ray diffraction of the crystal.
  • the present invention relates to techniques for determining the structure of RRF mutants, homologs, and the like, and for developing next-generation antibacterial agents, antifungal agents, and herbicides by applying the structural information and mechanism of action of RRF proteins.
  • Protein biosynthesis is an essential function of all cell life activities and consists of four stages: “start”, “extension”, “termination”, and “ribosome recycling”.
  • the final step in protein biosynthesis (the fourth step) is to release the messenger—termination RNA, transfer RNA, and ribosome termination complexes, respectively, to reuse the ribosomes for the next “start” step. It ends by dissociation.
  • Escherichia coli which is a prokaryotic organism
  • this “reuse” of ribosomes is based on ribosome recycling factor (RRF) and elongation factor G (EFG) or release factor 3 (Release factory). It is known to be catalyzed by tor3).
  • RRF ribosome recycling factor
  • EGF elongation factor G
  • Release factory release factor 3
  • HIV protease is crystallized and its three-dimensional structure is known. This structure and the three-dimensional structure of the active site Based on the amino acid sequence, a compound with the highest affinity for this site was selected from compounds known from computers and its inhibitory activity was measured. By forming a co-crystal of the target protein with the active one and measuring the three-dimensional structure, it is possible to predict the compound that binds more, and this is synthesized and its inhibitory activity is measured. Then, a co-crystal of this substance and the target protein is formed again, and an extremely effective substance can be obtained by repeating the above process.
  • the present inventors have determined several gene sequences for E. coli, not only for prokaryotes, but also for eukaryotes (Japanese Unexamined Patent Publication No. 3-20079). 7, PCT / JP98 / 00734, Japanese Patent Application No. 10-150493). Therefore, the secondary structure can be estimated from the amino acid sequence obtained therefrom.
  • the current state of the art it has not been possible to identify the actual three-dimensional structure from this secondary structure.
  • each amino acid residue interacts and, in some cases, undergoes various modifications to form its steric structure. Therefore, if the three-dimensional structure of a protein is known, it is possible to create a substance that can serve as a ligand. In order to create a useful antibiotic in this sense, determination of the three-dimensional structure by crystallization is extremely important. Will have significant significance.
  • an object of the present invention is to elucidate the three-dimensional structure of RRF and to contribute to the development of various antibacterial agents, antifungal agents and herbicides.
  • FIG. 1 is a photograph showing an XRRF protein crystal.
  • FIG. 2 is a photograph showing an X-ray diffraction image of an XRRF protein crystal.
  • FIG. 3 is a photograph of an RRF drawn with a ribbon. As shown in the figure, it consists of two domains, one consisting of three helical forces, and the second domain is a complex of ⁇ _sheet and coil helix.
  • FIG. 4 is a photograph of an RRF space filling model.
  • FIG. 5 is a schematic explanatory view showing a hypothesis about the mechanism of action of RRF.
  • FIG. 6 is a graph showing that various inhibitors inhibit release of transfer RNA from the termination complex. Error bars indicate standard deviation.
  • FIG. 7 Lineweaver—Burkf showing inhibition of liposome release in the presence of various concentrations of transferred RNII. This is a graph by lot.
  • FIG. 8 shows that RRF was transferred to ribosomes in the presence of paromomycin. It is a graph which shows that binding is inhibited. Error bars indicate standard deviation.
  • ribosome 1 ... ribosome, 2 ... transferred RNA, 3 ... messenger RNA, 4 ..- RRF, 5-EFG, 6 ... termination complex.
  • the present inventors have succeeded in obtaining RRF crystals and identifying the three-dimensional structure for the first time while conducting research on RRF, and as a result of further research, completed the present invention Reached.
  • the present invention relates to a crystal of R RF protein, its production method and three-dimensional structure.
  • a method for designing a compound capable of binding to the active site or an auxiliary binding site of an RRF protein, wherein the chemical entity is evaluated by computer based on the structural coordinates obtained from the RRF protein crystal relates to the method.
  • the present invention also relates to the above method, wherein the RRF protein crystal is any one of a crystal of the RRF protein itself, a crystal of an RRF protein mutant, a crystal of an RRF protein homolog, and a crystal of a co-complex of the RRF protein.
  • the present invention also relates to the above method, wherein the RRF protein crystal is a bipyramid system.
  • the present invention relates to a method in which the RRF protein crystal is a space group. 4,2,2, or have a space group P4 3 2, 2, 2, relates to the aforementioned method.
  • the present invention also relates to the above method, wherein the RRF protein crystal has a size of 0.3X0.3X0.5 mm.
  • the present invention relates to the aforementioned method, wherein the RRF protein crystal is characterized by the structural coordinates according to Table 7.
  • the present invention also relates to the above method, wherein the RRF protein crystal is derived from Thermotoga Martinima.
  • the present invention also relates to the above method, wherein the RRF protein crystal is orthorhombic. Furthermore, the present invention relates to the above method, wherein the RRF protein crystal has a space group P2, 2, 2. Further, the present invention relates to the above method, wherein the RRF protein crystal has a size of 30 3050 ⁇ 250 ⁇ 1.
  • the present invention also relates to the above method, wherein the RRF protein crystal is derived from bacterium X.
  • the present invention relates to the above method, wherein the RRF protein crystal is crystallized by a droplet vapor diffusion method.
  • the present invention also relates to the present invention, wherein the RRF protein crystal is a heavy atom derivative, and the crystal is any one of a crystal of the RRF protein itself, a crystal of the RRF protein mutant, a crystal of the RRF protein homolog, and a crystal of a co-complex of the RRF protein.
  • the method relates to the method.
  • the present invention also relates to the above method, wherein the heavy atom derivative is formed by reaction with a compound selected from the group consisting of thimerosal, gold thiomalate, peranyl acetate, and lead chloride.
  • the present invention relates to the above method, wherein the RRF protein crystal is a heavy atom derivative of platinum or mercury.
  • the present invention also relates to the above method, wherein the RRF protein is a monomer.
  • the present invention relates to the above method, characterized by amino acid displacement according to Table 5 or Table 6 of RRF protein strength.
  • the present invention relates to the above method, wherein the compound characterized by a chemical entity binding to an active site or an auxiliary binding site is an inhibitor of RRF protein.
  • the present invention relates to the method, wherein the inhibitor is a competitive, non-competitive or uncompetitive inhibitor of RRF.
  • the present invention also relates to the above method, comprising determining the orientation of the ligand at the active or auxiliary binding site of the RRF protein.
  • the present invention relates to the above method, wherein the structural coordinates are the structural coordinates of the RRF protein according to Table 7.
  • the present invention also relates to the above-mentioned method, wherein the RRF protein is a pocket near the C-terminus located at a bent portion separating two domains of the RRF protein. Furthermore, the present invention relates to the above method, wherein the compound inhibits binding of RRF protein to ribosome or inhibits behavior of RRF protein on liposome.
  • the present invention also relates to an RRF protein inhibitor obtained by the method.
  • the present invention also relates to an activity of inhibiting the binding of RRF protein to ribosome or an activity of inhibiting the behavior of RRF protein on ribosome.
  • the present invention relates to a method for searching for a compound that can inhibit the activity of an RRF protein based on the above.
  • the present invention relates to an RRF protein inhibitor obtained by the above method.
  • the present invention also relates to a method for determining the three-dimensional structure of the RRF protein, including elucidating the crystal form of the mutant, homolog or co-complex of the RRF protein by molecular replacement.
  • the invention also relates to orthorhombic RRF protein crystals.
  • the present invention also relates to the RRF protein crystal having a space group 1 ⁇ 2,2,2. Further, the present invention relates to the RRF protein crystal having a size of 30 ⁇ 50 ⁇ 250 ⁇ m.
  • the present invention also relates to the RRF protein crystal, wherein the RRF is derived from bacterium X.
  • the present invention also relates to a bipyramid-based RRF protein crystal.
  • the present invention relates to a space group?
  • the present invention relates to the RRF protein crystal having 4, 2, 2, or a space group of 4 3 2,2.
  • the present invention also relates to the RRF protein crystal having a size of 0.3X0.3X0.5mm.
  • the present invention also relates to the aforementioned RRF protein crystals, characterized by the amino acid changes according to Table 5 or Table 6.
  • the present invention is characterized in that the RRF tank is characterized by structural coordinates according to Table 7. Related to Park crystal.
  • the present invention also relates to the RRF protein crystal, which is derived from Thermotoga Multima.
  • the present invention also relates to the RRF protein crystal, which has been crystallized by a droplet vapor diffusion method.
  • the present invention relates to the RRF protein crystal, which is any one of a crystal of RRF protein itself, a crystal of an RRF protein mutant, a crystal of an RRF protein homologue, and a crystal of a co-complex of the RRF protein.
  • the present invention also relates to the RRF protein, wherein the amino acid at the active site is selected from the group consisting of Arg110, Arg129 and Arg132 of SEQ ID NO: 1.
  • the invention also provides that the one or more amino acids in the active site or the auxiliary active site are one or more amino acids selected from the group consisting of naturally occurring amino acids, unnatural amino acids, selenocystine and selenomethionine.
  • the RRF protein has been replaced by:
  • the present invention relates to the RRF protein, wherein the hydrophilic amino acid and the hydrophobic amino acid in the active site or the auxiliary active site are substituted.
  • the present invention also relates to the RRF protein, wherein at least one cysteine amino acid is substituted with an amino acid selected from the group consisting of selenocystine or selenomethionine.
  • the present invention also relates to the aforementioned RRF protein, wherein at least one methionine amino acid is substituted by an amino acid selected from the group consisting of selenocystine or selenomethionine.
  • the present invention relates to the RRF protein, which is in a crystalline form.
  • the present invention also relates to the RRF protein having a specific activity higher or lower than that of the wild-type enzyme.
  • the present invention also relates to the RRF protein having an altered substrate specificity.
  • the invention further relates to the use of said RRF protein for measuring the binding interaction between a compound and the RRF protein.
  • the present invention provides a method for treating at least 1 The RRF protein of any of the preceding claims, wherein one or more amino acid residues have been substituted, resulting in a change in one or more charge units of surface charge.
  • RRF is an ideal target of antibacterial agents
  • the three-dimensional structure of RRF disclosed by the present invention is extremely important in industry because it is directly linked to the development of antibacterial agents and the like.
  • the primary structure of RRF of many pathogens is very similar (for example, the RRF of Pseudomonas aeruginosa has 60% homology with that of Escherichia coli)
  • the three-dimensional structure of the RRF according to the present invention is These data make it very easy to understand the three-dimensional structure of the RRF of other pathogens.
  • the present invention is applied to the development of a next-generation antibiotic, an antifungal agent and a disinfectant by inhibiting RRF, and particularly to the development of an antibacterial agent by rational derag design. It is extremely useful as an indicator.
  • RRF protein means an RRF protein that has enzymatic activity under normal conditions.
  • Naturally occurring amino acid means the L-isomer of a naturally occurring amino acid.
  • Naturally occurring amino acids include glycine, alanine, valin, leucine, isoleucine, serine, methionine, threonine, fenylalanine, tyrosine, tryptophan, cystine, proline, histidine, and aspanolaginate.
  • the amino acids in the present specification are in the form of chow.
  • Unnatural amino acid refers to an amino acid that is not found in nature in a protein.
  • unnatural amino acids used herein include racemic mixtures of selenocystine and selenomethionine.
  • non-natural amino acids there are nor-mouth isine, para-nitrophenylalanine, homophenylalanine, nora-funoleolophenylalanine, and 3-amino-2-benzylpropionic acid. And D or L-form of homoarginine and D-phenylalanine.
  • “Positively charged amino acid” includes any naturally occurring or unnatural amino acid having a positively charged side chain under normal physiological conditions.
  • Examples of positively charged natural amino acids include arginine, lysine and histidine.
  • Negatively charged amino acid includes any naturally occurring or unnatural amino acid having a negatively charged side chain under normal physiological conditions.
  • negatively charged natural amino acids include aspartic acid and glutamic acid.
  • hydrophobic amino acid is meant any amino acid having an uncharged, non-polar side chain that is relatively insoluble in water.
  • hydrophobic amino acids are alanine, leucine, isoleucine, valin, proline, phenylalanine
  • Hydrophilic amino acid means any amino acid having an uncharged polar side chain that is relatively soluble in water.
  • hydrophilic amino acids are serine, threonine, tyrosine, asparagine, glutamine and cysteine.
  • Variant refers to an RRF polypeptide characterized by at least one amino acid substitution in the RRF sequence of wild-type E. coli (ie, a polypeptide that exhibits the biological activity of wild-type RRF).
  • variants can be obtained, for example, by expression of cDNA for RRF mutated in its coding sequence by oligonucleotide-specific induction.
  • RRF mutants can be obtained by general biosynthetic methods according to Noren, CJ, etc. (Science, 224, pl82-188 (1989)) by site-specific incorporation of unnatural amino acids into RRF proteins. be able to.
  • Selenocystin or selenomethionine is incorporated into wild-type or mutant RRF by expression of a cDNA encoding the RRF in an auxotrophic E. coli strain.
  • the wild-type or mutant RRF cDNA does not contain the power of natural cysteine or natural methionine (or both) and is grown on a growth medium enriched for selenocystine or selenomethionine (or both). Can be expressed in different hosts.
  • selenomethionine can be incorporated into wild-type or mutant RRF by the method of inhibiting methionine metabolism (J. B. by Van Dyne GD, 229 ppl05 (1993)).
  • change in surface charge is meant a change in one or more charge units of a variant polypeptide at physiological pH compared to wild-type RRF. This can preferably be obtained by mutation of at least one or more wild-type RRFs into amino acids containing side chains having a different charge from the wild-type side chain at physiological pH in the amino acid. The change in surface charge is determined by measuring the isoelectric point of the polypeptide with the substituted amino acid and comparing this with the isoelectric point of the wild-type RRF molecule.
  • “Change in substrate specificity” refers to a change in the substrate of a mutant RRF as compared to a wild-type RRF.
  • Substrate specificity is determined by separating ribosomes, tRNAs, and EF-G from pathogenic bacteria and determining whether they can serve as substrates for RRF and RRF variants of E. coli.
  • Kermic form refers to the state of the enzyme in free or unbound form or the state of the enzyme bound to a chemical entity at either its active site or ancillary active site.
  • a “competitive” inhibitor is one that inhibits RRF activity by binding to the same kinetic form of the RRF as the substrate of the RRF binds, and thus directly competes with the active site of the RRF.
  • “Uncompetitive” inhibitors are inhibitors that inhibit RRF by binding to a different kinetic form of RRF than the substrate binds to.
  • a “non-competitive” inhibitor is an inhibitor that binds to either the free or substrate-bound form of RRF.
  • homolog is meant a protein having at least 30% homology of the amino acid sequence with RRF or any functional domain of RRF.
  • co-complex is meant a RRF or a variant or homologue of an RRF covalently or non-covalently linked to a chemical entity or compound.
  • “/ 3-sheet” refers to the conformation of a polypeptide chain that extends into an expanded zigzag conformation. All parallel polypeptide chains extend in the same direction. Polypeptide chains extending antiparallel extend in the opposite direction to the parallel lines.
  • the substrate binding site is the site where the ribosome and its complex bind, and the site where degradation of the substrate occurs.
  • the active site is at least near amino acid residues 110, 129 and 132 using SEQ ID NO: 1. It is.
  • “Structural coordinates” refers to mathematical coordinates obtained from mathematical expressions relating to the pattern obtained by diffraction of an X-ray monochromatic beam by atoms (dispersion centers) of RRF molecules in a crystalline form. The variance data is used to calculate an electron density map of the repeating unit of the crystal, and the electron density map is used to establish the position of each atom within the unit cell of the crystal.
  • Heavy atom derivative refers to a chemically modified form of an RRF protein crystal.
  • heavy metal atom salts or organometallic compounds eg, lead chloride, gold thiomaleate, thyromesal or peranil acetate
  • the position (s) of the bound heavy metal atom (s) can be determined by X-ray diffraction analysis of the immersed crystal. This information is then used to create the phase information used to construct the three-dimensional structure of the enzyme. It will be appreciated by those skilled in the art that the set of structural coordinates determined by X-ray crystallography has a standard error.
  • a "unit cell” is a basic parallelepiped shaped block.
  • the total volume of the crystal can be constructed by repeated regular stacking of such blocks.
  • Space group refers to the arrangement of target elements of a crystal.
  • “Molecular substitution” means that the structural coordinates (for example, the structural coordinates in Table 7) of another unknown crystal are known in the unit cell of the unknown crystal so as to be optimal for explaining the observed diffraction pattern of the unknown crystal. It refers to a method that includes the step of orienting and positioning a molecule to create a tentative model of an RRF crystal whose structural coordinates are not known. Then the phase is calculated from this model and combined with the observed amplitude, An approximate Fourier composition of a structure whose coordinates are not known is obtained. It can then be applied to the purified material to finally obtain the exact unknown crystal structure.
  • the structural coordinates of the RRF it is possible to determine the structural coordinates of a mutant, homolog, co-complex or different crystal structure of the RRF by using molecular replacement. Crystallization and structural analysis were performed using RRF derived from X bacterium and RRF derived from Thermotoga Mitima, but the same can be performed for other RRFs. In crystallization, not only the RRF protein itself, but also RRF protein mutants, RRF protein homologs, and RRF protein co-complexes can be crystallized and their structures analyzed.
  • Pocket refers to a dent on the surface of the RRF protein, which binds to a substrate or the like in the expression of RRF activity, in addition to a binding pocket present in the binding site of the RRF protein or an auxiliary binding site. Include other pockets that are not involved in
  • the present invention provides, for the first time, a crystal of RRF of X bacterium and Thermotoga Maritima RRF and a structure of the RRF determined from the crystal.
  • Table 7 shows the structural coordinates of the RRF.
  • the crystal backing indicates that the RRF is monomeric.
  • Figure 3 shows a ribbon drawing of the Thermotoga Maritima RRF.
  • Helixes A, B, D, E, and F indicate a helix present from the N-terminus to the C-terminus.
  • ⁇ -sheets 1, 2, 3, 4, 5, and 6 are / 3-sheet numbers that exist from the ⁇ ⁇ ⁇ end to the C end.
  • the RRF is composed of two domains, one is composed of three helices, and the second domain is a complex of ⁇ -sheet coil and helix.
  • the active site spans the E and F helices in the figure, and maintains the three-dimensional structure of the domain containing helices B, C, D, / 3-sheets 1, 2, 3, 4, and 5 to maintain activity. Is important.
  • Figure 4 shows the space filling model of the Thermotoga Maritima RRF, where N and C are Shows the N-terminal and C-terminal, respectively. Gray indicates carbon, red indicates oxygen, purple indicates N atom, numbers indicate amino acid sequence number, and 1 is N-terminal.
  • the active site portion contains at least the amino acid residues Arg110, Arg129, and Arg132 of SEQ ID NO: 1.
  • the present invention enables, for the first time, the use of molecular design techniques to design, select and synthesize chemical entities and compounds with respect to RRF.
  • Chemical entities and compounds include inhibitory compounds that can bind to all or a portion of the active or auxiliary binding site of the RRF.
  • the structural coordinates of the RRF are used to design compounds that bind to the enzyme and to modify the physical properties (eg, solubility) of the compound in various ways. You.
  • the present invention allows for the design of compounds that act as competitive inhibitors of RRF by binding to all or a portion of the active site of the RRF.
  • the present invention also allows for the design of compounds that act as uncompetitive inhibitors of RRF.
  • inhibitors can bind to all or a portion of the auxiliary binding site of the RRF already bound to the substrate, and are more potent and more potent than competitive inhibitors, which only bind to the RRF active site. Can be non-specific. Similarly, non-competitive inhibitors that bind to and inhibit RRF, whether or not bound to another chemical entity, can be designed using the structure coordinates obtained according to the present invention.
  • a second design approach is to identify RRF crystals with molecules of various chemical entities in order to determine the optimal site for interaction between a candidate RRF inhibitor and RRF. For example, high-resolution X-ray diffraction data collected from solvent-saturated crystals allows the location of each type of solvent molecule to be determined. Small molecules that bind tightly to these sites can then be designed and synthesized, and tested for inhibitor activity (Travis, J., Science, 262, pl374 (1993)).
  • the present invention also provides for the reaction of a substrate or other compound that binds to the RRF with the RRF. It is useful in the design of improved analogs of RF inhibitors or in the design of new classes of inhibitors based on reaction intermediates of RRF and RRF inhibitor co-complexes. This provides a novel tool for designing RRF inhibitors with both high specificity and high stability.
  • Another approach enabled and facilitated by the present invention is the computer screening of chemical entities or compounds that can be wholly or partially bound to the RRF.
  • the properties of the fit of such an entity or compound to the binding site can be determined either by shape complementarity or by estimated interaction energies (Meng, EC et al. J. Comp. Chem. , 13, 505-524 (1992)).
  • the structural coordinates of the RRF, or portions thereof, as provided by the present invention will analyze the structure of other crystalline forms of the RRF Especially important for.
  • the structural coordinates of the RRF, or a portion thereof may be the structure of the RRF variant, the structure of the RRF co-complex, or the crystalline form of any other protein having an amino acid sequence that is significantly homologous to any functional domain of the RRF. Can also be used to analyze the structure.
  • the unknown crystal structure may be a crystal form of another form of RRF, an RRF variant or RRF co-complex or any other protein having an amino acid sequence that is significantly homologous to any functional domain of RRF.
  • RRF the structural coordinates of the RRF of the present invention as provided in Table 7. This method provides an accurate structural morphology for an unknown crystal more quickly and efficiently than trying to determine such information from scratch.
  • the RRF variant can be crystallized in a co-complex with a known RRF inhibitor.
  • the crystal structure of such complexes can then be solved by molecular replacement and compared to the crystal structure of wild-type RRF.
  • potential sites for modification within the various binding sites of the enzyme can be identified.
  • a means to determine the most effective binding interactions (eg, increased hydrophobic interactions) between the RRF and the chemical entity or compound I will provide a.
  • the structural coordinates of the RRF provided herein also facilitate the identification of related proteins, enzymes or M that are similar in function, structure, or both, to the RRF. Active sites such as serial similar protein, can more accurately estimate the binding sites and the like, a new antimicrobial agent, that connected to herbicides or antifungal agents.
  • Non-covalent intermolecular interactions that are important for the binding of RRF to its substrate include hydrogen bonding, van der Waals forces, and hydrophobic interactions.
  • the compound must be able to assume a conformation that allows it to bind to the RRF. Certain portions of the compound are not directly involved in binding to this RRF, but those portions can still affect the entire conformation of the molecule. This also has a significant effect on efficacy.
  • the prerequisites for such a conformation include the chemical entity or the entire three-dimensional structure and orientation of the compound for all or some of the binding sites (eg, the active or auxiliary binding site of the RRF), or the RRF. Examples include the spacing between functional groups of a compound that contains some chemical entity that interacts directly.
  • the potential inhibitory or binding effect of a chemical compound on RRF can be analyzed before it is actually synthesized, and using computer modeling techniques. Can be used for testing. If the theoretical structure of a given compound indicates that there is insufficient interaction and binding between that compound and RRF, the synthesis and testing of that compound can be avoided. However, if computer modeling suggests a strong interaction, the molecule is synthesized, and the method of Hirashima and Kaji (Bi ochemi stry, ⁇ , 4037, (1972)) or using oligonucleotides and in vivo Can be tested for its ability to inhibit by screening in Japan (Japanese Patent Application No. 10-158643). By this method, synthesis of ineffective compounds can be avoided.
  • Inhibitory compounds of RRF or other binding compounds of RRF can be evaluated computationally and chemical entities or fragments screened for their ability to bind to individual binding pockets or other regions of RRF. It can be designed by means of cleaning and a series of selected steps.
  • a chemical entity or fragment may be referred to as an RRF, more specifically, an individual binding pocket of a binding site or an auxiliary binding site of an RRF, or another pocket that is not involved in binding to a substrate or the like in expressing RRF activity.
  • RRF chemical entity or fragment
  • One of several ways to screen for their ability to combine with can be used. This process can be started by visual examination of active sites, for example, during computer screening based on the RRF coordinates in Table 7. For example, as shown in the drawing by the ribbon in Fig. 3, the RRF with an “L” shape has a pocket near the C-terminal located at the “L” bend that separates the two domains. However, compounds that bind to this pocket can be potential candidates for RRF inhibitory compounds.
  • the above-mentioned pockets can be easily observed by creating a space filling model using software such as Rasmol based on the RRF coordinates shown in Table 7. This pocket is located between the two domains of RRF, suggesting that it may be involved in the activity of RRF through regulation of the angle between the domains.
  • the selected fragments or chemical entities can then be located in various orientations or can be linked to individual binding pockets of the RRF. Coupling can be accomplished using software such as Quanta and Sybyl, and then using standard molecular mechanics force fields (eg, CHARMM, AMBER). Perform energy minimization and molecular dynamics.
  • Specialized computer programs can assist in the process of selecting fragments or chemical entities. Examples of these programs include:
  • uRID uoodiora, P. j., A omputat lonal Procedure for Determining tnerget ically Favorable Binding Sites on Biologically Important Mac romolecules ", J. Med. Chem., 28, pp. 849-857 (1985)) Is available from Oxford University, oxford, UK MCSS (Miranker, A and M, Karplus, "Functionality Map of Binding Sites: A Multiple Copy Simultaneous Search Method., Proteins-Structure, Function and Genetics, 11, pp. 29-34 (1991)), which is available from Molecular Simulations, Burlington, MA. AUT0D0CK (Goodsell, DS and ⁇ J.
  • CAVEAT Bartlett, PA et al, "CAVEAT: A Program to Facilitate the Structure-Derived Design of Biologiccal ly Active Molecules", Molle cular Recognition in Chemical and Biological Problems ", Royal Chem. So, 78, pp. 182-196 (1989)), which is the University of Carifornia,
  • MACCS 3D (3D Database systems such as MDL Information Systems, San Diego, CA; this area is described in Martin, Y. C., "3D Database Searching in Drug Design", J. Med. Chem., 35, pp. 2 145-2154 (1992) H00K (available from Molecular Simulations, Burlington, MA).
  • the inhibitory compound or other RRF-binding compound may have an active site (or, if necessary, known) from the RRF. (Including some portions of the inhibitors).
  • LUDI Bohm, HJ, "The Computer Program LUDI: A New Method for the de novo Design of Enzyme Inhibitors", J. Comp, Aid, Molec, Design, 6 pp. 61-78 (1992), which is Biosym Technologies, Available from San Diego, CA LEGEND (Nishibata, Y. and A. Itai, Tetrahedron, 47, p. 8985 (1991), available from Molecular Simulations, Burlington, MA. LeapFrog ( Tripos Associates, St. Louis, MO Power, etc. Available Ht.
  • the effectiveness with which the compound can bind to the RRF can be tested by computer evaluation and optimized.
  • a compound designed or selected to function as an RRF inhibitor depending on the active site when binding to a natural substrate Capacity that does not overlap with the capacity occupied should preferably be considered.
  • An effective RRF inhibitor should preferably show a relatively small difference in the energy between its bound and free states (ie, small binding strain forces. Therefore, the most effective RRF inhibitors
  • the RRF inhibitor should be designed with a binding strain of no more than about 10 kcal / mol, preferably no more than about 7 kcal / mol. In their conformation, they can interact with the enzyme, in which case the strain force of the bond is between the energy of the free compound and the average energy of the conformation observed when the inhibitor binds to the enzyme. Makes a difference.
  • Compounds designed or selected to bind to RRF, in their bound state, are preferably optimized by the computer to have no repulsive electrostatic interaction with the target enzyme.
  • Such non-complementary (eg, electrostatic) interactions repel, charge-charge, dipole-dipole, and charge-dipole interactions.
  • the sum of all electrostatic interactions between the inhibitor and the enzyme when the inhibitor binds to RRF makes a neutral or favorable contribution to the enthalpy of binding.
  • Certain computer software is available in the art to evaluate compound strain forces and electrostatic interactions. Examples of programs designed for such uses include Gaussian 92C, MJ Frisch, Gaussian, Inc., Pittsburgh, PA 1992; AMBER, version 4.0 PA Kollman, University of California, San Francisco, 1994; QUANTA / CHARMM Molecular Simulat ions, Inc, San Diego, CA 1994 and the like. These programs can be executed using a general-purpose computer such as Silicon Graphics IRIS 4d / 35 or IBM RISC / 6000 Model550. Other hardware and software are known to those skilled in the art.
  • substitutions are then made on some of the atoms or side chains of the compound to improve or modify its binding properties.
  • the first substitution is conservative. That is, the substituent has approximately the same size, shape, hydrophobicity, and charge as the original group. In the field Compounds known to alter the conformation should be avoided.
  • the chemical compounds so substituted are then analyzed for potency compatible with the RRF in a manner similar to the computational methods described above.
  • the present invention also allows for variants of RRF and elucidation of their crystal structure. More specifically, the present invention allows the identification of the desired site for mutation by the location of the active site, auxiliary binding site and interface of the RRF based on the crystal structure of the RRF.
  • the mutation can be directed to a particular site or combination of only the wild-type RRF site, ie, the active site or the auxiliary binding site.
  • a location on the interface site is selected for mutagenesis.
  • only positions on or near the enzyme surface can be replaced, resulting in a change in the surface charge of one or more charged units as compared to the wild-type enzyme.
  • the amino acid residues of the RRF can be selected based on their hydrophilic or hydrophobic characteristics.
  • Such variants are characterized by any of several different properties as compared to the wild-type RRF.
  • a variant may have a change in the surface charge of one or more charged units, or may have increased stability to subunit dissociation.
  • such a mutant may have an altered substrate specificity as compared to the wild-type RRF, or may have a higher or lower specific activity than the wild-type RRF.
  • the RRF variants prepared according to the present invention can be prepared by a number of methods.
  • a wild-type RRF sequence can be mutated using the present invention at the site identified as desirable for mutation by oligonucleotide-directed mutagenesis or other conventional techniques (eg, deletion, etc.).
  • variants of RRF can be created by site-specific substitution of a particular amino acid with a non-naturally occurring amino acid.
  • RRF variants can be made by replacing amino acid residues, ie, specific cysteine or methionine residues, with selenocysteine or selenomethionine.
  • Mutations can be introduced into the DNA sequence encoding the RRF using synthetic oligonucleotides. These oligonucleotides contain nucleotide sequences flanking the desired mutation site. Mutations can be made in the full length DNA sequence of the RRF, or the RRF or the RRF sequence of other organisms, shortened or lengthened (deleted or added).
  • mutated RRF DNA sequences produced by the methods described above or alternative methods known in the art can be expressed using expression vectors.
  • expression vectors typically include an element that enables autonomous replication in the host cell independent of the host genome, and one or more phenotypic markers for selection purposes.
  • the expression vector also encodes a promoter, an operator, a ribosome binding site, a translation initiation signal, and, optionally, a libresor gene and a termination signal, before or after the insert of the DNA sequence surrounding the desired RRF variant coding sequence. Contains regulatory sequences.
  • a nucleotide coding for a signal sequence can be inserted before the RRF mutant code sequence.
  • the desired DNA sequence must be operably linked to the regulatory sequences. That is, a DNA sequence encoding the RRF variant and maintaining an appropriate reading frame that allows expression of this sequence under the control of a regulatory sequence and production of the desired product encoded by the RRF sequence. Must have an appropriate start signal before the.
  • a wide variety of well-known and available expression vectors are all useful for expressing the mutated RRF coding sequences of the present invention. These include, for example, various known derivatives of SV40, known bacterial plasmids (eg, plasmids from E. coli including colEl, pCR1, pBR322, pMB9 and derivatives thereof).
  • Plasmids of wider host range eg, RP4, phage DNA (eg, derivatives of many phage (eg, NM989) and other DNA phages (eg, M13 and filamentous single-stranded DNA phage)
  • 2 Yeast plasmids such as ⁇ plasmid or their derivatives and plasmids and It should consist of segments of chromosomal, non-chromosomal and synthetic DNA sequences, such as vectors obtained from combinations of phage DNAs (eg, phage DNA or plasmids modified to utilize other expression control sequences). Includes turtles. In a preferred embodiment of the present invention, we utilize the E. coli vector.
  • any of a wide variety of expression control sequences that control expression when operably linked to a DNA sequence may be used in these vectors to express a mutated DNA sequence according to the present invention.
  • useful expression control sequences include, for example, the early and late promoters of SV40 for animal cells, the lac, trp, TAC or TRC systems, and the ⁇ phage regulatory region of the fd coat protein.
  • Major operator region and promoter region all for E.
  • a wide variety of host species are also useful for producing mutant RRFs according to the present invention.
  • these hosts include bacteria such as E. coli, Bacillus and Streptomyces, fungi such as yeast, animal cells such as CH0 cells and COS-1 cells, plant cells, and transgenics.
  • bacteria such as E. coli, Bacillus and Streptomyces, fungi such as yeast, animal cells such as CH0 cells and COS-1 cells, plant cells, and transgenics.
  • Host cells In a preferred embodiment, the host cell is E. coli.
  • the host must be compatible with the selected vector, have the toxicity of the modified RRF to the host, have the ability to secrete mature products, have the ability to properly fold the protein, have the fermentation requirements, have the ease of purifying the modified RRF from the host, Should be selected based on safety considerations.
  • one of skill in the art can select various vector / expression control Z host combinations that can produce useful amounts of mutant RRF.
  • Mutant RRF produced in these systems can be purified by a variety of conventional processes and strategies, including those used to purify wild-type RRF and strategies.
  • the mutant can be tested for any of several properties of interest.
  • mutants can be screened for changes in charge at physiological pH. This is determined by measuring the isoelectric point of the mutant RRF compared to the isoelectric point (pi) of the wild-type parent. The isoelectric point is determined by gel electrophoresis according to the method of Wellner, D. Analyt. Chem. 43. P 597 (1971). Variants with altered surface charge are RRF polypeptides having a substituted amino acid located on the surface of the enzyme and an altered pi, as provided by the structural information of the present invention.
  • variants can be screened for higher or lower specific activity compared to wild-type RRF. Mutants are measured for activity using the method of Hirashima and Kaji and Atsushi (supra) using oligonucleotides. Mutants can be tested for changes in RRF substrate specificity by measuring the RRF response as described above.
  • a further object of the present invention includes variants with increased stability.
  • RRF variants with increased stability include those that do not show loss of enzyme activity.
  • Example 1 Crystallization of RRF protein of fungus X by the hanging drop vapor ditt'usion technique
  • the glass container with which the RRF protein solution comes into contact is used after its surface is subjected to a hydrophobic treatment.
  • XRRF crystals were obtained by dialysis against a buffer solution of Tris hydrochloride 100 mM ⁇ 8.5 sulfate 150 mM to 200 mM, polyethylene dalicol 28% to 36%. The crystals grew to a size of 30X50X250 im in one to three weeks. Figure 1 shows the results.
  • Example 2 Three-dimensional structure of RRF by X-ray diffraction analysis
  • a multiple isomorphous replacement procedure was used as a means for determining the three-dimensional structure of the RRF. This is the standard method necessary to obtain diffusion data from isotopic protein crystals due to heavy atoms. From the position of the heavy atom, the difference between the unsubstituted isotope and the isotope was calculated on a Patterson map. The data of the initial protein phase necessary for the calculation of the electron density map for the creation of the protein model was calculated using several types of derivatives.
  • This asymmetric unit belonging to 2 contains 2 to 4 molecules, with 0.5, 0.33, and 0.5 translations between each molecule. Data were obtained for two derivatives, the platinum derivative diffracted to 4.OA and the mercury derivative to 3.8A.
  • the RRF cDNA of Thermotoga Maritima was cloned into an expression vector (PET1650) and expressed in E. coli by adding IPTG.
  • PTT1650 expression vector
  • high levels of Thermotog a Maritima RRF accumulated in host cells.
  • the cells were mechanically disrupted and purified by a modification of the method of Hirashima and Kaji (Biochemistry, ⁇ , 4037, (1972)) to obtain Thermotoga Maritima RRF.
  • RRF crystals were grown by vapor diffusion.
  • the position of the selenium atom in the RRF was determined by using the shelx program (sheldrick, GM Acta Cryst. A46 P467 (1998)), and using the normalized structure factor. Heavy atom (selenium) parameters were refined using MIphase (CCP4). When I asked the electron density map in both the space group 1 ⁇ 4,2,2 and ⁇ 4 3 2,2, space group correct, it has become clear that it is ⁇ 42,2. Average merit values ranged from 0.66 0.6 to 4.OA. Table 2 shows the crystal data of Thermotoga Maritima RRF. Table 2 Data collection
  • ⁇ 1> is the average intensity of centrally symmetric reflection.
  • Table 3 shows the results of the statistical processing of the Thermotoga Maritima RRF crystal data.
  • RRF variants were generated to deduce the location of the active site in the RRF molecule. Mutagenesis was introduced using an error-prone PCR method (Janosi et al EMBO J. 17 1141 (1998)).
  • Plasmid having frr (gene encoding RRF) having a lethal mutation was isolated as follows. The pMIX described in EMBO J. 17 1141 (1998) by Janosi et al. Was used. Briefly, pMIX caused various genetic mutations in frr and introduced them into chloramphenicol-resistant plasmid. In this example, E. coli LJ4 (recA_) was used as a host. Escherichia coli is kept alive by the wild-type frr on pPEN (1560) (Janosi et al. EMBO JU 1141 (1998)) because this fungus is inactivated by frr on the chromosome due to frameshifting. . pPEN (1560) has a kanamycin resistance factor and sucrose ill sucrose sensitivity gene 's'.
  • the E. coli was transformed with pMIX and selected for chloramphenicol resistance as a marker. Since frr is indispensable to bacteria, bacteria having a lethal mutation in pMIX cannot survive without pPEN1560. Therefore, bacteria having both pMIX and PPEN1560 plasmids were searched. By the way, both pMIX and PPEN1560 plasmids do not coexist because they are usually incompatible, but coexist when required (need for antibiotic marker and frr) as described above. To select such Escherichia coli, the transformant is spread on a plate containing CM and sucrose, and then replica-plated on a plate containing CM and KM. To go.
  • bacteria that have lethal frr in pPEN1560 and pMIX can be selected. Since this bacterium has pPEN1560, it cannot grow on plates containing sucrose. Each plasmid was purified from the 153 transformants thus obtained, and used to transform Escherichia coli DH5 (having wild type frr). Since this bacterium has a wild-type frr as described above, it does not require pPEN1560 (kanamycin resistance). Therefore, by selecting Escherichia coli DH5a which is sensitive to kuram ramphenicol and kanamycin, Escherichia coli having lethal mutation and having pMIX can be selected.
  • Plasmid was isolated from the Escherichia coli thus obtained, and a Kpnl-Hindlll fragment (0.9 kb, frr) was taken out and subjected to DNA sequencing by a conventional method. Table 5 shows the results.
  • LJ4 was used as a host for this purpose. This host can be present at 27 ° C due to the plasmid pKH6 with frrl4 as frr on the chromosome does not function as described above.
  • This Escherichia coli is naturally temperature-sensitive because it lives by frr14 (encoding a temperature-sensitive RRF). Which grow the E. coli 42 ° C at a rate of the growing 4.2x 10- 6 Letting and 2 7 ° C the natural reversion rate was obtained. Among these Escherichia coli, those that became temperature-sensitive again when plasmid was replaced with one having tsfrr (pKH6) were selected, and the DNA sequence of the frr portion was determined by a conventional method. In all of the obtained frr, the genetic mutation of frr, Val 117 Asp, was reverted to wild-type palin, but some of them showed mutations at amino acid positions other than position 117. These mutations had no effect on frr function. This mutation is shown in Table 6.
  • RRF a mutation that restores the temperature sensitivity of a gene
  • NA —, W1-wild cattle type
  • tS temperature sensitivity
  • tr temperature tolerance
  • RRF4 binds to the aminoacyl site (A site) of the termination complex 6 (a) consisting of transfer RNAs 2a and 2b, messenger RNA 3 and ribosome 1.
  • EFF5 with GTP is bound to RRF4.
  • Transfer RNAs 2a and 2b are bound to the peptidyl site (P site) and the release site (exit site) (E site) of ribosome 1, respectively (b).
  • EFG5 ribosome-dependent GTP hydrolysis and translocation of the RRF4 bound to the A site to the P site is triggered by EFG5.
  • Aminoglycosides such as streptomycin, paromomycin, and gentamicin are known to inhibit the binding of translocated RNA to the A site by binding to the A site of the ribosome (Moazed, D. & Noller, HF Nature 327, 389-394 (1987); Fourmy, D., Yoshizawa, S. & Puglisi, JDJ Mol. Biol. 277, 333-345 (1988)); Yoshizawa, S., Fourmy, D. & Puglisi, JD EMBO J.17, 64 37-6448 (1988)). Therefore, according to the model in FIG.
  • the aminoglycosides should also inhibit the binding of RRF to the A site, and if such binding is inhibited, the dissociation process of the termination complex by RRF will be inhibited. Should be. Therefore, whether or not the dissociation process of the termination complex is inhibited in the presence of the above aminodaricosides was examined using the amount of transfer RNA released from liposomes and the amount of liposome released from messenger RNA as indicators. Was.
  • the released transfer RNA was separated from the termination complex by centrifugation at 330 G for 40 minutes using Microcon 100 (trade name, manufactured by Millipore).
  • Knocker J tris-CI 10 mM, pH 7.6, magnesium sulfate 10 mM, ammonium chloride 50 mM, DTT 0.5 mM was added to the above Microcon 100.
  • the filter was washed once by pouring 550 ⁇ l and centrifuging.
  • the combined washing solution and filtrate were centrifuged twice at 1,500 G for 15 minutes using Microc o ⁇ 30 (Mil 1 ipore, trade name) to obtain 1 It was concentrated to 41.
  • the concentrated transfer RNA was added to 30 ⁇ l of buffer solution (Tris_C 150 mM, pH 7.8, magnesium acetate 10 mM, 3_mercaptoethanol 6 mM, ATP 3 mM, phosphoenoenoside).
  • Monorubic acid 5 mM, pyruvate kinase 1 38 ⁇ g, aminosyltransferred RNA synthase 33.3 ⁇ g (Momose, K. & Kaji, A. Arch. Biochem. Biophys. Ill. 245-252 (1965))), a mixture of 14 C-amino acids (Amersham, 52 mCi / mg carbon atom) dissolved in 0.15 Ci And aminoacylated.
  • the radioactivity insoluble in cold trichloroacetic acid (4 ° C) obtained in this way corresponds to 14 C-aminoacyl-transferred RNA, and its amount is determined by a known amount of transfer RNA labeled by the same method. Was calculated based on the radioactivity.
  • As aminoglycosides those manufactured by Sigma were used.
  • the termination complex was obtained from a natural polysome of E. coli treated with puromycin (Hirashima, A. & Kaji, AJ Biol. Chem. 248, 7580-7587 (1973)).
  • each ribosome in the isolated polysome is in a stage after the completion of translocation and generally carries two molecules of transfer RNA (Remme, J., Margus, T., Villems, R. & Nierhaus, KH Eur. J. Biochem. 183, 28 g 284 (1989); Stark, H. et al. Cell 88, 19-28 (1977)).
  • Fig. 6 shows the results.
  • the positive control is the value when RRF, EFG and GTP were added without adding the aminoglycosides
  • the negative control was the aminoglycosides, RRF, £ ⁇ and 0 Are the values when none of the above was added.
  • aminoglycosides streptomycin, paromomycin or gentamicin were added to give 100, 5 and 5 ⁇ 5, respectively. Next, 15 to 30 in buffer J. /. The above buffer solution was incubated on 5 ml of sucrose with a density gradient, and the mixture was incubated at 4 ° C, 75 minutes, 4 ° C using Beckman SW 50.1. Centrifuge. Absorbance at 254 nm
  • Table 7 shows the results.
  • the values in Table 7 are the percentages of the concentration of 70S liposome released in the presence of aminoglycosides and the concentration of free 70S ribosome without aminoglycosides (control). It is expressed in the context. In the control, about 42% of all ribosomes (almost 90% of polysomes) were converted to monosomes by RRF. Table 7 By RRF and EF-G in the presence of various inhibitors
  • Thiostrepton and biomycin are both EFG inhibitors and are known to inhibit the translocation of transfer RNA (Pestka, S.B. iochem. Biophys. Res. Comraun. 40, 667-674 (1970); Rodnina, MV, Savel sbergh, A., Katunin, VI & Wintermeyer, W. Nature 385, 37-41 (1997); Rodn ina, MV et al. Pro Natl. Acad. Sci. USA 96, 9586-9590 (1999)).
  • transfer RNA Pestka, S.B. iochem. Biophys. Res. Comraun. 40, 667-674 (1970); Rodnina, MV, Savel sbergh, A., Katunin, VI & Wintermeyer, W. Nature 385, 37-41 (1997); Rodn ina, MV et al. Pro Natl. Acad. Sci. USA 96, 9586-9590 (1999)).
  • RNA from the P and E sites that should occur, and thus the dissociation process of the termination complex by RRF should also be inhibited. Therefore, whether or not the dissociation process of the termination complex is inhibited in the presence of thiostrepton or biomycin is determined by measuring the amount of transfer RNA released from liposomes and the amount of liposomes released from messenger RNA. I investigated.
  • RNA from ribosomes Release of RNA was examined in the same manner as in Example 4, except that thiostrepton (Sigma) or biomycin (ICN) was added instead of amidglycosides.
  • Thiostrepton and biomycin were added at 100 ⁇ M and 200 / X M, respectively.
  • Figure 6 shows the results.
  • the release of ribosomes from messenger RNA can be achieved by adding DMSO to the reaction mixture at the point where thiostrepton or biomycin is added instead of amide glycosides and when thiostrepton is added. Except for the addition, it was examined in the same manner as in Example 4. Thiostrepton and biomycin were added at 20 ⁇ M and 50 ⁇ M, respectively. Table 7 shows the results.
  • FIG. 6 shows that in the presence of thiostrepton or biomycin, the release of metastatic RNA was inhibited to a value equivalent to that of the negative control.
  • Table 7 shows that the release of ribosome is completely inhibited in the presence of thiostrepton or biomycin.
  • GMP PCP and fusidic acid are both EFG inhibitors and are terminated by immobilizing EFG on liposomes after translocation of transfer RNA It is known to inhibit the dissociation of RNA, while allowing translocation of transfer RNA only once (Inoue-Yokosawa, N., Ishikawa, C. & Kaziro, YJ Biol. Chem. 249, 4321-4323 (1974); Rodnina, MV, Savelsberg, A., Katunin, VI & Wintermeyer, W. Nature 385, 37-41 (1997); Bodley, J. W., Zieve, FJ, Lin, L.
  • GMP PCP and fusidic acid allow translocation of the RRF bound to the A site to the P site, so that GMP PCP and fusidic acid are released from the P site and E site that accompany the translocation. Transfer RNA release should not be inhibited.
  • GMP PCP and fusidic acid should inhibit the dissociation of the ribosome from messenger RNA because they inhibit the dissociation of the termination complex. Therefore, it was examined whether or not the release of transfer RNA from ribosomes and the release of liposomes from messenger RNA were inhibited in the presence of GMP PCP or fusidic acid.
  • Transfer from ribosome RNA is released by adding GMP PCP (manufactured by Sigma) or fusidic acid (manufactured by ICN) instead of amidoglycosides and without GTP when GMP PCP is added.
  • GMP PCP manufactured by Sigma
  • fusidic acid manufactured by ICN
  • ribosomes from messenger RNA also depends on the fact that GMP PCP or fusidic acid was added instead of amide glycosides, and that when GMP PCP was added, experiments were performed in the absence of GTP. Except for this, the procedure was the same as in Example 4. GMP PCP or fusidic acid was added so as to be 3700 ⁇ M and 200 ⁇ M, respectively. Table 7 shows the results.
  • RRF may exhibit termination complex dissociation activity by exhibiting behavior similar to that of transfer RNA.Furthermore, the release of transfer RNA and ribosome release are separately inhibited. The fact that can be done supports the model shown in FIG. 5, in which the dissociation of the termination complex proceeds stepwise by EFG and RRF.
  • Example 4 strongly suggested that the expression of RRF activity required RRF to bind to the A site of the ribosome. Therefore, if excess transfer RNA is present, RRF and transfer RNA will compete and bind to the A site of the liposome, which should inhibit dissociation of the termination complex. To confirm this point, the inhibition of dissociation of the termination complex in the presence of various concentrations of transfer RNA was examined.
  • Polysome (0. 5 ⁇ 1 A 26. Units) a, 0 to 1 000 pmo various amount between le RRF, EFG, transfer of various amounts between GTP and 0 ⁇ 1 0 nmo 1 e Incubated in the presence of RNA. Then, after performing sucrose density gradient centrifugation (density gradient: 15 to 30%), the amount of free ribosome was measured by measuring the absorbance at 254 nm. Lineweavwe—Burk created based on the measurement results. Figure 7 shows a graph based on the lot. The vertical axis is the reciprocal of the percentage of free ribosome in the presence of various amounts of transfer RNA relative to the amount of free ribosome in the absence of transfer RNA.
  • Example 4 Based on the results of and example 7, in order to directly verify the paromomycin used in Example 4. inhibit the binding of liposomes RRF, was first subjected to the following experiments, 35 of less than 1 pmo 1 e S-labeled histidine tag RRF to (35 S- H is -RR F) , in the presence of Paromo Maishishin of 1 0 pmo 1 e washed ribosomes and various concentrations of buffer (g squirrel - CI 5 0mM, p H 7 6. Incubated at 30 ° C for 10 minutes in 10 mM magnesium acetate, 30 mM potassium chloride, 1 mM DTT).
  • buffer g squirrel - CI 5 0mM, p H 7 6.
  • 35 S—His—RRF was prepared by purifying His—RRF expressed in vitro in the presence of 35 S-labeled methionine using Ni 2+ beads.
  • Fig. 8 shows the results.
  • the RRF binding ratio is a value calculated by assuming that the amount of RRF bound to ribosomes in the absence of paromomycin is 100%.
  • FIG. 8 shows that the binding rate of RRF decreases depending on the concentration of paromomycin. Therefore, paromomycin is thought to inhibit the activity of RRF by inhibiting the binding of RRF to ribosomes.
  • Atom type residue ## XYZ OCC B Atom 1 CB VA then 2 10.355 24.444 73.500 1.00 50.36 Atom 2 CGI VA then 2 11.185 25.300 72.669 1.00 50.36 Atom 3 CG2 VA 2 9.102 25.267 74.125 1.00 50.36 Atom 4 C VAL 2 8.502 23.777 72.304 1.00 83,11 atoms 50 VA then 2 8.267 24.906 71.890 1.00 83.11 atoms 6 N VA then 2. 10.415 23.206 71.242 1.00 83.11 ⁇

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Abstract

L'invention concerne une méthode de conception d'un composé capable de se lier au site actif ou au site de liaison accessoire d'une protéine du facteur de recyclage ribosomique (RRF), la méthode consistant à évaluer par ordinateur le corps chimique d'une protéine de RRF sur la base des coordonnées de structure provenant de cristaux protéine de RRF ; et une méthode de recherche d'un composé capable d'inhiber l'activité d'une protéine de RRF sur la base de l'activité d'inhibition de la liaison d'une protéine de RRF à un ribosome ou d'une activité d'inhibition du comportement d'une protéine de RRF sur un ribosome. Ces méthodes s'utilisent pour clarifier la structure tridimensionnelle et le mécanisme de fonctionnement du RRF, ce qui contribue à l'élaboration de plusieurs bactéricides, fongicides, herbicides, etc.
PCT/JP2000/003639 1999-06-04 2000-06-05 Cristal de proteine du facteur de recyclage ribosomique (rrf) et son application sur la base de donnees de structure tridimensionnelles provenant du cristal WO2000075182A1 (fr)

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JP2005512954A (ja) * 2001-07-12 2005-05-12 ベーリンガー インゲルハイム インターナショナル ゲゼルシャフト ミット ベシュレンクテル ハフツング ヒトパピローマウイルスｅ２トランスアクチベーションドメイン／インヒビター共結晶および前記インヒビター結合ポケットの境界を定めるｘ線座標

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JP2005512954A (ja) * 2001-07-12 2005-05-12 ベーリンガー インゲルハイム インターナショナル ゲゼルシャフト ミット ベシュレンクテル ハフツング ヒトパピローマウイルスｅ２トランスアクチベーションドメイン／インヒビター共結晶および前記インヒビター結合ポケットの境界を定めるｘ線座標

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