WO2005001417A2 - Cristaux de la polymerase de l'hepatite c et/ou des proteines de type polymerase du virus de l'hepatite c et utilisation associee - Google Patents

Cristaux de la polymerase de l'hepatite c et/ou des proteines de type polymerase du virus de l'hepatite c et utilisation associee Download PDF

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WO2005001417A2
WO2005001417A2 PCT/US2004/016763 US2004016763W WO2005001417A2 WO 2005001417 A2 WO2005001417 A2 WO 2005001417A2 US 2004016763 W US2004016763 W US 2004016763W WO 2005001417 A2 WO2005001417 A2 WO 2005001417A2
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hepatitis
virus polymerase
arg
leu
lys
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WO2005001417A3 (fr
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Barry C. Finzel
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Pharmacia & Upjohn Company
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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
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B15/00—ICT specially adapted for analysing two-dimensional or three-dimensional molecular structures, e.g. structural or functional relations or structure alignment
    • G16B15/30—Drug targeting using structural data; Docking or binding prediction
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1241—Nucleotidyltransferases (2.7.7)
    • C12N9/127—RNA-directed RNA polymerase (2.7.7.48), i.e. RNA replicase
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/576—Immunoassay; Biospecific binding assay; Materials therefor for hepatitis
    • G01N33/5767—Immunoassay; Biospecific binding assay; Materials therefor for hepatitis non-A, non-B hepatitis
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B15/00—ICT specially adapted for analysing two-dimensional or three-dimensional molecular structures, e.g. structural or functional relations or structure alignment
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2299/00—Coordinates from 3D structures of peptides, e.g. proteins or enzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/24011—Flaviviridae
    • C12N2770/24211—Hepacivirus, e.g. hepatitis C virus, hepatitis G virus
    • C12N2770/24222—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/91—Transferases (2.)
    • G01N2333/912—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • G01N2333/91205—Phosphotransferases in general
    • G01N2333/91245—Nucleotidyltransferases (2.7.7)
    • G01N2333/9125—Nucleotidyltransferases (2.7.7) with a definite EC number (2.7.7.-)

Definitions

  • This invention relates to the crystallization and structure determination of hepatitis C virus polymerase, also known as HCV polymerase or HCV.
  • HCV hepatitis C virus
  • N-structural protein 5b (NS5b) of the virus has been identified as an RNA- dependent RNA polymerase that must catalyze viral RNA (+)-strand synthesis during replication. A method of identifying inhibitors of HCV polymerase is needed in the art.
  • the present invention provides a crystal of hepatitis C virus polymerase, and methods of crystallizing a hepatitis C virus polymerase, the crystal having trigonal space group symmetry P3 2 21.
  • the hepatitis C virus polymerase is a monomer and the crystal includes one monomer per asymmetric unit.
  • the hepatitis C virus polymerase has an amino acid sequence including SEQ ID NO: 1.
  • the hepatitis C virus polymerase has an amino acid sequence including SEQ ID NO: 1 , with the proviso that a methionine is replaced with selenomethionine. Methods of using such crystals are also provided.
  • the present invention provides a method of preparing a crystal of hepatitis C virus polymerase complex including exposing a crystal of hepatitis C virus polymerase having trigonal space group symmetry P3 2 21 to a fluid including a potential modifier.
  • the present invention provides a method of acquiring structural information for designing potential modifiers for forming molecular complexes with hepatitis C virus polymerase.
  • the method includes: exposing a crystal of hepatitis C virus polymerase having trigonal space group symmetry P3 21 to a library of potential modifiers having diverse shapes; and determining whether a potential modifier-hepatitis C virus polymerase molecular complex is formed.
  • the method further includes identifying the chemical entity that forms the potential modifier upon determination of the formation of the potential modifier-hepatitis C virus polymerase molecular complex.
  • determining and/or identifying includes calculating an electron density function and/or collecting x-ray diffraction data.
  • the present invention provides a molecule or molecular complex having trigonal space group symmetry P3 2 21 including at least a portion of a hepatitis C virus polymerase or hepatitis C virus polymerase-like binding site, wherein the binding site includes the amino acids listed in Table 4, Table 5, or Table 6, and the binding site is defined by a set of points having a root mean square deviation ol less than about 0.65 A from points representing the backbone atoms of said amino acids as represented by the structure coordinates listed in Table 1 , Table 2, or Table 3.
  • the present invention provides a molecular complex including at least a portion of a hepatitis C virus polymerase or hepatitis C virus polymerase-like binding site, wherein the binding site includes the amino acids listed in Table 4 and the binding site is defined by a set of points having a root mean square deviation of less than about 0.65 A from points representing the backbone atoms of said amino acids as represented by the structure coordinates listed in Table 2 or Table 3.
  • the present invention provides a molecule or molecular complex having trigonal space group symmetry P3 2 21 that is structurally homologous to a hepatitis C virus polymerase molecule or molecular complex, wherein the hepatitis C virus polymerase molecule or molecular complex is represented by at least a portion of the structure coordinates listed in Table 1 , Table 2, or Table 3.
  • the present invention provides a molecular complex that is structurally homologous to a hepatitis C virus polymerase molecular complex, wherein the hepatitis C virus polymerase molecular complex is represented by at least a portion of the structure coordinates listed in Table 2 or Table 3.
  • the present invention provides a scalable three- dimensional configuration of points, at least a portion of said points derived from structure coordinates as listed in Table 2 or Table 3 of at least a portion of a hepatitis C virus polymerase molecule and at least a portion of a potential modifier, the configuration of points including a hepatitis C virus polymerase or hepatitis C virus polymerase-like binding site.
  • at least a portion of the points derived from the hepatitis C virus polymerase structure coordinates are derived from structure coordinates representing the locations of at least the backbone atoms of amino acids defining a hepatitis C virus polymerase binding site including the amino acids listed in Table 4, Table 5, or Table 6.
  • the three-dimensional configuration of points is displayed as a holographic image, a stereodiagram, a model, or a computer-displayed image.
  • the present invention provides a scalable three- dimensional configuration of points, at least a portion of the points derived from structure coordinates of (i) at least a portion of a molecule that is structurally homologous to a hepatitis C virus polymerase molecule or molecular complex and includes a hepatitis C virus polymerase or hepatitis C virus polymerase-like binding site, and (2) at least a portion of a potential modifier.
  • the present invention provides a machine-readable data storage medium including a data storage material encoded with a first set of machine readable data which, when combined with a second set of machine readable data, using a machine programmed with instructions for using said first set of data and said second set of data, can determine at least a portion of the structure coordinates corresponding to the second set of machine readable data, wherein said first set of data includes a Fourier transform of at least a portion of the structural coordinates as listed in Table 1 , Table 2, or Table 3 for a hepatitis C virus polymerase molecule or molecular complex having trigonal space group symmetry P3 2 21, and said second set of data includes an x-ray diffraction pattern of a molecule or molecular complex of unknown structure.
  • the present invention provides a machine-readable data storage medium including a data storage material encoded with a first set of machine readable data which, when combined with a second set of machine readable data, using a machine programmed with instructions for using said first set of data and said second set of data, can determine at least a portion of the structure coordinates corresponding to the second set of machine readable data, wherein said first set of data includes a Fourier transform of at least a portion of the structural coordinates as listed in Table 2 or Table 3 for a hepatitis C virus polymerase molecular complex, and said second set of data includes an x-ray diffraction pattern of a molecule or molecular complex of unknown structure.
  • the present invention provides a method for obtaining structural information about a molecular complex of unknown structure.
  • the method includes: crystallizing the molecular complex; generating an x-ray diffraction pattern from the crystallized molecular complex; and applying to the x-ray diffraction pattern at least a portion of the structure coordinates as set forth in Table 2 or Table 3 for hepatitis C virus polymerase to generate a three- dimensional electron density map of at least a portion of the molecular complex whose structure is unknown.
  • the present invention provides a method for homology modeling a hepatitis C virus polymerase homolog.
  • the method includes: aligning the amino acid sequence of a hepatitis C virus polymerase homolog with an amino acid sequence of hepatitis C virus polymerase and incorporating the sequence of the hepatitis C virus polymerase homolog into a model of hepatitis C vims polymerase formed from structure coordinates as set forth in Table 1, Table 2, or Table 3 for hepatitis C virus polymerase to yield a preliminary model of the hepatitis C virus polymerase homolog; subjecting the preliminary model to energy minimization to yield an energy minimized model; and remodeling regions of the energy minimized model where stereochemistry restraints are violated to yield a final model of the hepatitis C virus polymerase homolog.
  • the present invention provides computer-assisted methods for identifying, designing, or making a potential modifier of hepatitis C virus polymerase activity.
  • the methods include screening a library of chemical entities.
  • HCV Hepatitis C virus
  • HCVpol Hepatitis C virus polymerase
  • Nonstructural Protein 5b (NS5b) 4-(2-Hydroxyethyl)-l-piperazineethanesulfonic acid (HEPES)
  • Ethylenediaminetetraacetic acid EDTA
  • Root mean square (r. .s.)
  • Figure 1 is an illustration of the amino acid sequence (SEQ ID NO: 1) of hepatitis C virus I B J l C-delta-21.
  • Figure 2 is an illustration of the chemical structures of potential modifiers.
  • Figure 2a is the chemical structure of PHA-562769.
  • Figure 2b is the chemical structure of PHA-729145.
  • Figure 3 is a ribbon representation of the structure of HCV NS5b polymerase.
  • the location of the product grip hai ⁇ in e.g., residues 443-454
  • the location of the primer grip e.g., residues 362-368) is inside the circle.
  • Figures 3a and 3b are two roughly pe ⁇ endicular views.
  • Figure 4 is a ribbon representation of the structure of HCV NS5b polymerase illustrating the core Primer Grip binding site.
  • Two bound compounds e.g., PHA-562769 and PHA-729145, the locations of which are indicated by arrows
  • Figures 4a and 4b are two roughly pe ⁇ endicular views.
  • Figure 5 illustrates the relationship between bound PHA-562769 and bound PHA-729145, the locations of which are indicated by arrows, in the Primer Grip binding site.
  • a surface is shown to illustrate pocket geometry created by neighboring enzyme amino acid residues.
  • Figure 6 is an illustration of the amino acid sequence of HCV genotype lb, isolate Jl full-length protein (SEQ ID NO:2).
  • Figure 7 is an illustration of the three-dimensional structure of the HCV polymerase having three distinct domains identified as Fingers, Palm, and Thumb, after the polymerase nomenclature of Kohlstaedt et al., Science, 256: 1783-1790 (1992).
  • the connection segment to the C-terminal hydrophobic domain of NS5b is the gray strand indicated by the arrow.
  • the hydrophobic domain black on the bottom of the sidebar
  • the final 7 residues (564-570) are disordered in the structure and so omitted from the figure.
  • This figure was prepared with software Molscript v2.0 (Kraulis, J. Appl.
  • Tables 1-3 list atomic structure coordinates derived by x-ray diffraction for crystals of native hepatitis C virus polymerase, crystals of PHA-562769- hepatitis C virus polymerase, and PHA-729145-hepatitis C virus polymerase, respectively. Column 1 lists a number for the atom in the structure. Column 2 lists the element whose coordinates are measured. The first letter(s) in the column define the element.
  • column 3 lists the type of amino acid using the three-letter abbreviations listed herein above; if the element is part of a water molecule, column 3 lists HOH; if the element is part of a potential modifier or inhibitor, column 3 lists INH; if the element is part of a phosphate anion, column 3 lists PO4; if the element is part of a bound glycerol, column 3 lists GOL; and if the element is a chloride anion, column 3 lists CL1.
  • Column 4 lists the chain id (A for the inhibitor-HCVpol molecular complex in the asymmetric unit, B for chloride and/or phosphate anions, C for bound glycerols, and W for water molecules).
  • Column 5 lists a number for the amino acid or molecule in the structure.
  • Columns 6-8 list the crystallographic coordinates X, Y, and Z respectively. The crystallographic coordinates define the atomic position of the element measured.
  • Column 9 lists an occupancy factor that refers to the fraction of the molecules in which each atom occupies the position specified by the coordinates. A value of " 1 " indicates that each atom has the same conformation, i.e., the same position, in all molecules of the crystal.
  • Column 10 lists a thermal factor "B” that measures movement of the atom around its atomic center.
  • hepatitis C virus polymerase is from the HCV genotype IB J l strain. More preferably, the hepatitis C virus polymerase is from truncated HCV genotype IB J l strain (C-delta-21).
  • the crystal has trigonal space group symmetry P3 2 21.
  • the crystallized enzyme is a monomer having six monomers in the unit cell (i.e., one monomer per asymmetric unit).
  • Crystals can be prepared from frozen HCV NS5b protein in buffer containing 20 mM Tris pH 7.5, 20% glycerol, 500 mM NaCl, 10 mM MgCl 2 , 250 mM imidazole, 0.1 mM PMSF, 2 ⁇ g/ml leupeptin and 5 mM ⁇ ME at a protein concentration of 1.5-2.5 mg/ml.
  • the P3 2 21 crystal form of hepatitis C virus polymerase may be used with soaking methods to form molecular complexes with potential modifiers.
  • “native” form means that the crystallized hepatitis C virus polymerase molecule does not include substantial amounts of binding sites having a potential modifier complexed thereto.
  • a “potential modifier” refers to a chemical entity that could function as a drug candidate (e.g., modifiers and inhibitors).
  • a "native" crystal includes at least about 70% uncomplexed binding sites, more preferably at least about 90% uncomplexed binding sites, even more preferably at least about 95% uncomplexed binding sites, and most preferably about 100% uncomplexed binding sites.
  • the native P3 2 21 crystal form of hepatitis C virus polymerase led to several experiments to test its utility in hepatitis C virus polymerase-potential modifier molecular complex formation.
  • a "molecular complex” means a protein in covalent or non-covalent association with a chemical entity (e.g., a potential modifier). Crystal forms that possess large enough solvent channels can be used to form complexes by soaking potential modifiers into the crystal.
  • Solid inhibitor can preferably be dissolved in DMSO to make 100 mM stock solution, or diluted to 50 mM if solubility is limited.
  • Inhibitor stock solutions may preferably be added to both soaking and cryo solutions to bring the concentration of inhibitor to 2 mM throughout soaking and cryoprotection.
  • the soaking solution with compound may preferably be added (slowly) directly to the drops over approximately 1-1.5 hours. Soaking solution may be added, for example, in the following volumes with approximately 15 minutes between additions: O.lul, 0.25ul, 0.5ul, l.Oul, 2.0ul, -2ul +2ul.
  • the transfers to cryo solutions may be done using the same additions as described for the soaking solution, with one added step. Before plunging the crystals into liquid nitrogen, the crystals may be dipped in 100% cryo solution for 30seconds.
  • the total soaking time is generally recorded at the beginning of the transfers and also includes the time during cryo solution transfers. Crystals are typically soaked for between 4 hours and 3 days. Routine use of the P3 2 21 crystal form for the preparation of hepatitis C virus polymerase-potential modifier complexes may significantly reduce the time between receipt of a compound for testing and generation of a complex. Because separate co-crystallization experiments are not required for each compound, complexes can be generated within a few days and subsequently data can be collected. Crystallography may preferably be used to screen and identify chemical entities that are not known potential modifiers of target biomolecules as disclosed, for example, in U.S. Pat. No. 6,297,021 (Nienaber et al.).
  • crystallography may preferably be used to screen and identify chemical entities that are not known potential modifiers of hepatitis C virus polymerase for their ability to bind to hepatitis C virus polymerase.
  • a preferred method includes obtaining a crystal of hepatitis C virus polymerase; exposing the hepatitis C virus polymerase to one or more test samples that include a potential modifier of the hepatitis C virus polymerase; and determining whether a potential modifier-hepatitis C virus polymerase molecular complex is formed.
  • the hepatitis C virus polymerase may be exposed to potential modifiers by various methods including, for example, soaking a hepatitis C virus polymerase crystal in a solution of one or more potential modifiers, or co-crystallizing hepatitis C virus polymerase in the presence of one or more potential modifiers.
  • Structural information from the potential modifier-hepatitis C virus polymerase complexes found may preferably be used to design new potential modifiers that bind tighter, bind more specifically, have desired biological activity properties, have better safety profiles than known potential modifiers, and combinations thereof.
  • libraries of "shape-diverse" chemical entities may preferably be used to allow direct identification of the potential modifier-hepatitis C virus polymerase complex even when the potential modifier is exposed as part of a mixture.
  • shape diverse refers to potential modifiers having substantial differences in three-dimensional shapes that can be recognized, for example, by visual inspection of the two dimensional chemical structures, or by calculation and comparison of relevant parameters by a computational program. Shape diversity of the mixture permits a bound potential modifier to be identified directly from the resultant electron density map. This preferably avoids the need for time-consuming deconvolution of a hit from the mixture.
  • three important steps are preferably achieved simultaneously.
  • the calculated electron density function directly reveals the binding event, identifies the bound chemical entity, and provides a detailed 3-D structure of the potential modifier-hepatitis C virus polymerase complex.
  • a hit preferably a number of analogs or derivatives of the hit may be screened for tighter binding or desired biological activity by traditional screening methods.
  • the identity of the hit and information about structure of the target may preferably be used to develop analogs or derivatives with tighter binding or desired biological activity properties.
  • the potential modifier-hepatitis C virus polymerase complex may be exposed to additional iterations of potential modifiers so that two or more hits may preferably be linked together to identify or design a more potent potential modifier.
  • structure coordinates refers to Cartesian coordinates derived from mathematical equations related to the patterns obtained on diffraction of a monochromatic beam of x-rays by the atoms (scattering centers) of a hepatitis C virus polymerase complex in crystal form.
  • the diffraction data are used to calculate an electron density map of the repeating unit of the crystal.
  • the electron density maps are then used to establish the positions of the individual atoms of the hepatitis C virus polymerase protein or protein-potential modifier complex.
  • Slight variations in structure coordinates can be generated by mathematically manipulating the hepatitis C virus polymerase or hepatitis C virus polymerase-potential modifier structure coordinates.
  • the structure coordinates set forth in Table 1 could be manipulated by crystallographic permutations of the structure coordinates, fractionalization of the structure coordinates, integer additions or subtractions to sets of the structure coordinates, inversion of the structure coordinates or any combination of the above.
  • modifications in the crystal structure due to mutations, additions, substitutions, and/or deletions of amino acids, or other changes in any of the components that make up the crystal could also yield variations in structure coordinates. Such slight variations in the individual coordinates will have little effect on overall shape.
  • the resulting three- dimensional shape is considered to be structurally equivalent. Structural equivalence is described in more detail below. It should be noted that slight variations in individual structure coordinates of the hepatitis C virus polymerase would not be expected to significantly alter the nature of chemical entities such as potential modifiers that could associate with the binding sites.
  • the phrase "associating with” refers to a condition of proximity between a chemical entity, or portions thereof, and a hepatitis C virus polymerase molecule or portions thereof. The association may be non-covalent, wherein the juxtaposition is energetically favored by hydrogen bonding, van der Waals forces, or electrostatic interactions, or it may be covalent.
  • a potential modifier that bound to a binding site of hepatitis C virus polymerase would also be expected to bind to or interfere with a structurally equivalent binding site.
  • “residue” refers to one or more atoms.
  • Particularly preferred structurally equivalent molecules or molecular complexes are those that are defined by the entire set of structure coordinates listed in Table 1 ⁇ a root mean square deviation from the conserved backbone atoms of those amino acids of less than about 0.65 A. More preferably, the root mean square deviation is at most about
  • a molecular complex defined by the structure coordinates listed in Table 1 for those amino acids listed in Table 4, Table 5, or Table 6 ⁇ a root mean square deviation from the conserved backbone atoms of those amino acids of less than about 0.65 A, preferably at most about 0.5 A, and more preferably at most about 0.35 A.
  • root mean square deviation means the square root of the arithmetic mean of the squares of the deviations. It is a way to express the deviation or variation from a trend or object.
  • the "root mean square deviation" defines the variation in the backbone of a protein from the backbone of hepatitis C virus polymerase or a binding site portion thereof, as defined by the structure coordinates of hepatitis C virus polymerase described herein. It will be readily apparent to those of skill in the art that the numbering of amino acids in other isoforms of hepatitis C virus polymerase may be different than that of hepatitis C virus polymerase expressed in E. coli.
  • Binding sites are of significant utility in fields such as drug discovery.
  • the association of natural potential modifiers or substrates with the binding sites of their corresponding receptors or enzymes is the basis of many biological mechanisms of action.
  • many drugs exert their biological effects through association with the binding sites of receptors and enzymes. Such associations may occur with all or any parts of the binding site. An understanding of such associations helps lead to the design of drugs having more favorable associations with their target, and thus improved biological effects.
  • binding site refers to a region of a molecule or molecular complex, that, as a result of its shape, favorably associates with another chemical entity.
  • a binding site may include or consist of features such as cavities, surfaces, or interfaces between domains.
  • Chemical entities that may associate with a binding site include, but are not limited to, cofactors, substrates, modifiers, agonists, and antagonists.
  • the amino acid constituents of a hepatitis C virus polymerase binding site as defined herein are positioned in three dimensions in accordance with the structure coordinates listed in Table 1, Table 2, and/or Table 3.
  • the structure coordinates defining a binding site of hepatitis C virus polymerase include structure coordinates of all atoms in the constituent amino acids; in another aspect, the structure coordinates of a binding site include structure coordinates of just the backbone atoms of the constituent amino acids.
  • the binding site of hepatitis C virus polymerase preferably includes the amino acids listed in Table 4, more preferably the amino acids listed in Table 5, and most preferably the amino acids listed in Table 6, as represented by the structure coordinates listed in Table 1, Table 2, and/or Table 3.
  • the binding site of hepatitis C virus polymerase may be defined by those amino acids whose backbone atoms are situated within about 4 A, more preferably within about 7 A, most preferably within about 10 A, of one or more constituent atoms of a bound substrate or modifier.
  • the binding site may be defined by those amino acids whose atoms are situated within a given distance of atoms of a bound potential modifier (e.g., an inhibitor) as defined in Table 2 or Table 3, the distance being about 4 A, preferably about 7 A, and more preferably about 10 A.
  • hepatitis C virus polymerase-like binding site refers to a portion of a molecule or molecular complex whose shape is sufficiently similar to at least a portion of a binding site of hepatitis C virus polymerase as to be expected to bind related structural analogues.
  • at least a portion means that at least about 50% of the amino acids are included, preferably at least about 70% of the amino acids are included, more preferably at least about 90% of the amino acids are included, and most preferably all the amino acids are included.
  • a structurally equivalent binding site is defined by a root mean square deviation from the structure coordinates of the backbone atoms of the amino acids that make up binding sites in hepatitis C virus polymerase (as set forth in Table 1) of at most about 0.35 A. How this calculation is obtained is described below. Accordingly, the invention provides molecules or molecular complexes including a hepatitis C virus polymerase binding site or hepatitis C virus polymerase-like binding site, as defined by the sets of structure coordinates described above.
  • HCV polymerase (NS5b) was crystallized and the structure was determined.
  • a C-terminally truncated construct of the HCV-1 Jl strain (Jl C ⁇ 21) was crystallized in a unique trigonal crystal form (P3 2 21) with one molecule in the crystallographic asymmetric unit.
  • the sequence of the construct is given in Figure 6.
  • the structure was solved by molecular replacement, and was subsequently refined against diffraction data extending to 1.9 A resolution.
  • the final R-value is 0.198 (Table 7).
  • the final model includes all residues from the N-terminus to Ser-563.
  • the HCV NS5b structure has an architecture that has been compared to a right hand with Fingers, Palm and Thumb domains (Figure 7). This topology is shared with a variety of different DNA and RNA-dependent polymerases.
  • the Fingers of HCV polymerase are particularly large, dominated by extended beta structure that reaches all the way across the palm to interact extensively with the thumb, giving the impression of a somewhat closed hand.
  • One large loop inserted into the palm domain e.g., residues 228-283
  • a large globular Fingers domain has not been seen in other polymerase structures in the absence of template primer.
  • the thumb is very well developed also, and is larger and better ordered than in other viral polymerases (Hansen et al., Structure, 5: 1109-1122 ( 1997)).
  • a 34 residue segment at the end of the thumb would normally connect to the C-terminal hydrophobic domain truncated from the J1 ⁇ 21 construct.
  • This "connection segment” takes an extended and meandering course back across the front of the palm to interact with the beta hai ⁇ in of the thumb (e.g., residues 443-454). This segment cannot therefore be considered part of the thumb and has been colored gray in Figure 7.
  • Residues 22-28 and 149-153 are part of external loops on the back of the fingers the are largely disordered in all of the structures.
  • Residues 306-309 form a connection between helix ⁇ J and the catalytic beta sheet of the palm domain, but is far from the active site and on the molecular surface.
  • Residues 532-546 show some variability among the four available C ⁇ 21 structures. No large systematic differences in domain position are revealed by this comparison.
  • the binding site is adjacent to the "Primer Grip” motif (e.g., residues 363-367) (Hansen et al., Structure, 5:1109-1 122 (1997)), and so is called the "Primer Grip Binding site.”
  • the site is also bounded by another beta- hai ⁇ in called the “Substrate Grip” (e.g., residues 443-454) because of suspected role of this segment in gripping oligonucleotide duplex substrates (Lesburg et al., Nat. Struct. Biol, 6:937-943 (1999)).
  • the position of both of these segments is highlighted in the illustrations of the native structure depicted in Figure 3.
  • THREE-DIMENSIONAL CONFIGURATIONS X-ray structure coordinates define a unique configuration of points in space. Those of skill in the art understand that a set of structure coordinates for protein or an protein-potential modifier complex, or a portion thereof, define a relative set of points that, in turn, define a configuration in three dimensions. A similar or identical configuration can be defined by an entirely different set of coordinates, provided the distances and angles between coordinates remain essentially the same. In addition, a scalable configuration of points can be defined by increasing or decreasing the distances between coordinates by a scalar factor while keeping the angles essentially the same.
  • the present invention thus includes the scalable three-dimensional configuration of points derived from the structure coordinates of at least a portion of a hepatitis C virus polymerase molecule or molecular complex, as listed in Table 1 , as well as structurally equivalent configurations, as described below.
  • the scalable three-dimensional configuration includes points derived from structure coordinates representing the locations of a plurality of the amino acids defining a hepatitis C virus polymerase binding site.
  • the scalable three-dimensional configuration includes points derived from structure coordinates representing the locations the backbone atoms of a plurality of amino acids defining the hepatitis C virus polymerase binding site, preferably the amino acids listed in Table 4, more preferably the amino acids listed in Table 5, and most preferably the amino acids listed in Table 6.
  • the scalable three-dimensional configuration includes points derived from structure coordinates representing the locations of the side chain and the backbone atoms (other than hydrogens) of a plurality of the amino acids defining the hepatitis C virus polymerase binding site, preferably the amino acids listed in Table 4, more preferably the amino acids listed in Table 5, and most preferably the amino acids listed in Table 6.
  • the invention also includes the scalable three-dimensional configuration of points derived from structure coordinates of molecules or molecular complexes that are structurally homologous to hepatitis C virus polymerase, as well as structurally equivalent configurations. Structurally homologous molecules or molecular complexes are defined below.
  • structurally homologous molecules can be identified using the structure coordinates of hepatitis C virus polymerase according to a method of the invention.
  • the configurations of points in space derived from structure coordinates according to the invention can be visualized as, for example, a holographic image, a stereodiagram, a model, or a computer-displayed image, and the invention thus includes such images, diagrams or models.
  • STRUCTURALLY EQUIVALENT CRYSTAL STRUCTURES Various computational analyses can be used to determine whether a molecule or a binding site portion thereof is "structurally equivalent,” defined in terms of its three-dimensional structure, to all or part of hepatitis C virus polymerase or its binding sites. Such analyses may be carried out in current software applications, such as the Molecular Similarity application of QUANTA (Molecular Simulations Inc., San Diego, CA) version 4.1, and as described in the accompanying User's Guide. The Molecular Similarity application permits comparisons between different structures, different conformations of the same structure, and different parts of the same structure.
  • the procedure used in Molecular Similarity to compare structures is divided into four steps: (1) load the structures to be compared; (2) define the atom equivalences in these structures; (3) perform a fitting operation; and (4) analyze the results.
  • Each structure is identified by a name.
  • One structure is identified as the target (i.e., the fixed structure); all remaining structures are working structures (i.e., moving structures).
  • atom equivalency within QUANTA is defined by user input, for the purpose of this invention equivalent atoms are defined as protein backbone atoms (N, C ⁇ , C, and O) for all conserved residues between the two structures being compared.
  • a conserved residue is defined as a residue which is structurally or functionally equivalent. Only rigid fitting operations are considered.
  • the working structure is translated and rotated to obtain an optimum fit with the target structure.
  • the fitting operation uses an algorithm that computes the optimum translation and rotation to be applied to the moving structure, such that the root mean square difference of the fit over the specified pairs of equivalent atom is an absolute minimum. This number, given in angstroms, is reported by QUANTA.
  • the invention thus further provides a machine-readable storage medium including a data storage material encoded with machine readable data which, when using a machine programmed with instructions for using said data, displays a graphical three-dimensional representation of any of the molecule or molecular complexes of this invention that have been described above.
  • the machine-readable data storage medium includes a data storage material encoded with machine readable data which, when using a machine programmed with instructions for using said data, displays a graphical three- dimensional representation of a molecule or molecular complex including all or any parts of a hepatitis C virus polymerase binding site or an hepatitis C virus polymerase-like binding site, as defined above.
  • the machine-readable data storage medium includes a data storage material encoded with machine readable data which, when using a machine programmed with instructions for using said data, displays a graphical three- dimensional representation of a molecule or molecular complex defined by the structure coordinates of all of the amino acids listed in Table 1 , ⁇ a root mean square deviation from the backbone atoms of said amino acids of less than about 0.65 A, more preferably at most about 0.5 A, and even more preferably, at most about 0.35 A.
  • the machine-readable data storage medium includes a data storage material encoded with a first set of machine readable data which includes the Fourier transform of the structure coordinates set forth in Table 1, and which, when using a machine programmed with instructions for using said data, can be combined with a second set of machine readable data including the x-ray diffraction pattern of a molecule or molecular complex to determine at least a portion of the structure coordinates corresponding to the second set of machine readable data.
  • a system for reading a data storage medium may include a computer including a central processing unit (“CPU”), a working memory which may be, e.g., RAM (random access memory) or “core” memory, mass storage memory (such as one or more disk drives or CD-ROM drives), one or more display devices (e.g., cathode-ray tube (“CRT”) displays, light emitting diode (“LED”) displays, liquid crystal displays (“LCDs”), electroluminescent displays, vacuum fluorescent displays, field emission displays (“FEDs”), plasma displays, projection panels, etc.), one or more user input devices (e.g., keyboards, microphones, mice, track balls, touch pads, etc.), one or more input lines, and one or more output lines, all of which are interconnected by a conventional bidirectional system bus.
  • CPU central processing unit
  • working memory which may be, e.g., RAM (random access memory) or “core” memory
  • mass storage memory such as one or more disk drives or CD-ROM drives
  • display devices e
  • the system may be a stand-alone computer, or may be networked (e.g., through local area networks, wide area networks, intranets, extranets, or the internet) to other systems (e.g., computers, hosts, servers, etc.).
  • the system may also include additional computer controlled devices such as consumer electronics and appliances.
  • Input hardware may be coupled to the computer by input lines and may be implemented in a variety of ways. Machine-readable data of this invention may be inputted via the use of a modem or modems connected by a telephone line or dedicated data line. Alternatively or additionally, the input hardware may include CD-ROM drives or disk drives. In conjunction with a display terminal, a keyboard may also be used as an input device.
  • Output hardware may be coupled to the computer by output lines and may similarly be implemented by conventional devices.
  • the output hardware may include a display device for displaying a graphical representation of a binding site of this invention using a program such as QUANTA as described herein.
  • Output hardware might also include a printer, so that hard copy output may be produced, or a disk drive, to store system output for later use.
  • a CPU coordinates the use of the various input and output devices, coordinates data accesses from mass storage devices, accesses to and from working memory, and determines the sequence of data processing steps.
  • a number of programs may be used to process the machine-readable data of this invention. Such programs are discussed in reference to the computational methods of drug discovery as described herein.
  • Machine-readable storage devices useful in the present invention include, but are not limited to, magnetic devices, electrical devices, optical devices, and combinations thereof.
  • Examples of such data storage devices include, but are not limited to, hard disk devices, CD devices, digital video disk devices, floppy disk devices, removable hard disk devices, magneto-optic disk devices, magnetic tape devices, flash memory devices, bubble memory devices, holographic storage devices, and any other mass storage peripheral device.
  • these storage devices include necessary hardware (e.g., drives, controllers, power supplies, etc.) as well as any necessary media (e.g., disks, flash cards, etc.) to enable the storage of data.
  • the structure coordinates set forth in Table 1 can be used to aid in obtaining structural information about another crystallized molecule or molecular complex.
  • the method of the invention allows determination of at least a portion of the three-dimensional structure of molecules or molecular complexes which contain one or more structural features that are similar to structural features of hepatitis C virus polymerase. These molecules are referred to herein as "structurally homologous" to hepatitis C virus polymerase.
  • Similar structural features can include, for example, regions of amino acid identity, conserved active site or binding site motifs, and similarly arranged secondary structural elements (e.g., ⁇ helices and ⁇ sheets).
  • structural homology is determined by aligning the residues of the two amino acid sequences to optimize the number of identical amino acids along the lengths of their sequences; gaps in either or both sequences are permitted in making the alignment in order to optimize the number of identical amino acids, although the amino acids in each sequence must nonetheless remain in their proper order.
  • two amino acid sequences are compared using the Blastp program, version 2.0.9, of the BLAST 2 search algorithm, as described by Tatusova et al., FEMS Microbiol Lett., 174:247-50 (1999), and available on the world wide web at ncbi.nlm.nih.gov/gorf/bl2.html.
  • a structurally homologous molecule is a protein that has an amino acid sequence sharing at least 65% identity with a native or recombinant amino acid sequence of hepatitis C virus polymerase (for example, SEQ ID NO: 1). More preferably, a protein that is structurally homologous to hepatitis C virus polymerase includes a contiguous stretch of at least 50 amino acids that shares at least 80% amino acid sequence identity with the analogous portion of the native or recombinant hepatitis C virus polymerase (for example, SEQ ID NO: l).
  • this invention provides a method of utilizing molecular replacement to obtain structural information about a molecule or molecular complex whose structure is unknown including the steps of: (a) crystallizing the molecule or molecular complex of unknown structure; (b) generating an x-ray diffraction pattern from said crystallized molecule or molecular complex; and (c) applying at least a portion of the structure coordinates set forth in Table 1 to the x-ray diffraction pattern to generate a three-dimensional electron density map of the molecule or molecular complex whose structure is unknown.
  • hepatitis C virus polymerase or the hepatitis C virus polymerase-potential modifier complex as provided by this invention can be used to determine the structure of a crystallized molecule or molecular complex whose structure is unknown more quickly and efficiently than attempting to determine such information ab initio.
  • Molecular replacement provides an accurate estimation of the phases for an unknown structure. Phases are a factor in equations used to solve crystal structures that cannot be determined directly. Obtaining accurate values for the phases, by methods other than molecular replacement, is a time-consuming process that involves iterative cycles of approximations and refinements and greatly hinders the solution of crystal structures.
  • this method involves generating a preliminary model of a molecule or molecular complex whose structure coordinates are unknown, by orienting and positioning the relevant portion of hepatitis C virus polymerase or the hepatitis C virus polymerase/modifier complex within the unit cell of the crystal of the unknown molecule or molecular complex so as best to account for the observed x-ray diffraction pattern of the crystal of the molecule or molecular complex whose structure is unknown.
  • Phases can then be calculated from this model and combined with the observed x-ray diffraction pattern amplitudes to generate an electron density map of the structure whose coordinates are unknown.
  • This in turn, can be subjected to any well-known model building and structure refinement techniques to provide a final, accurate structure of the unknown crystallized molecule or molecular complex (Lattman, Meth. Enzymol, 1 15, 55-77 (1985); M.G. Rossman, ed., "The Molecular Replacement Method," Int. Sci. Rev. Ser., No. 13, Gordon & Breach, New York (1972)).
  • Structural information about a portion of any crystallized molecule or molecular complex that is sufficiently structurally homologous to a portion of hepatitis C virus polymerase can be resolved by this method.
  • a molecule that shares one or more structural features with hepatitis C virus polymerase as described above a molecule that has similar bioactivity, such as the same catalytic activity, substrate specificity, or potential modifier binding activity as hepatitis C virus polymerase, may also be sufficiently structurally homologous to hepatitis C virus polymerase to permit use of the structure coordinates of hepatitis C virus polymerase to solve its crystal structure.
  • the method of molecular replacement is utilized to obtain structural information about a molecule or molecular complex, wherein the molecule or molecular complex includes a hepatitis C virus polymerase subunit or homolog.
  • a "subunit" of hepatitis C virus polymerase is a hepatitis C virus polymerase molecule that has been truncated at the N-terminus or the C-terminus, or both.
  • a "homolog" of hepatitis C virus polymerase is a protein that contains one or more amino acid substitutions, deletions, additions, or rearrangements with respect to the amino acid sequence of hepatitis C virus polymerase (SEQ ID NO: l), but that, when folded into its native conformation, exhibits or is reasonably expected to exhibit at least a portion of the tertiary (three-dimensional) structure of hepatitis C virus polymerase.
  • structurally homologous molecules can contain deletions or additions of one or more contiguous or noncontiguous amino acids, such as a loop or a domain.
  • Structurally homologous molecules also include "modified" hepatitis C virus polymerase molecules that have been chemically or enzymatically derivatized at one or more constituent amino acid, including side chain modifications, backbone modifications, and N- and C- terminal modifications including acetylation, hydroxylation, methylation, amidation, and the attachment of carbohydrate or lipid moieties, cofactors, and the like.
  • a heavy atom derivative of hepatitis C virus polymerase is also included as a hepatitis C virus polymerase homolog.
  • the term "heavy atom derivative” refers to derivatives of hepatitis C virus polymerase produced by chemically modifying a crystal of hepatitis C virus polymerase.
  • a crystal is soaked in a solution containing heavy metal atom salts, or organometallic compounds, e.g., lead chloride, gold thiomalate, thiomersal or uranyl acetate, which can diffuse through the crystal and bind to the surface of the protein.
  • the location(s) of the bound heavy metal atom(s) can be determined by x-ray diffraction analysis of the soaked crystal. This information, in turn, is used to generate the phase information used to construct three-dimensional structure of the protein (T.L. Blundell and N.L. Johnson, Protein Crystallography, Academic Press ( 1976)).
  • the structure coordinates of hepatitis C virus polymerase as provided by this invention are particularly useful in solving the structure of other crystal forms of hepatitis C virus polymerase or hepatitis C virus polymerase complexes.
  • the structure coordinates of hepatitis C virus polymerase as provided by this invention are particularly useful in solving the structure of hepatitis C virus polymerase mutants. Mutants may be prepared, for example, by expression of hepatitis C virus polymerase cDNA previously altered in its coding sequence by oligonucleotide-directed mutagenesis.
  • Mutants may also be generated by site- specific inco ⁇ oration of unnatural amino acids into hepatitis C virus polymerase proteins using the general biosynthetic method of Noren et al., Science, 244:182- 88 (1989).
  • the codon encoding the amino acid of interest in wild- type hepatitis C virus polymerase is replaced by a "blank" nonsense codon, TAG, using oligonucleotide-directed mutagenesis.
  • a suppressor tRNA directed against this codon is then chemically aminoacylated in vitro with the desired unnatural amino acid.
  • the aminoacylated tRNA is then added to an in vitro translation system to yield a mutant hepatitis C virus polymerase with the site-specific inco ⁇ orated unnatural amino acid.
  • Selenocysteine or selenomethionine may be inco ⁇ orated into wild-type or mutant hepatitis C virus polymerase by expression of hepatitis C virus polymerase-encoding cDNAs in auxotrophic E. coli strains (Hendrickson et al., EMBO J., 9: 1665-72 (1990)).
  • the wild-type or mutagenized hepatitis C virus polymerase cDNA may be expressed in a host organism on a growth medium depleted of either natural cysteine or methionine (or both) but enriched in selenocysteine or selenomethionine (or both).
  • selenomethionine analogues may be prepared by down regulation methionine biosynthesis.
  • the structure coordinates of hepatitis C virus polymerase listed in Table 1 are also particularly useful to solve the structure of crystals of hepatitis C virus polymerase, hepatitis C virus polymerase mutants or hepatitis C virus polymerase homologs co-complexed with a variety of chemical entities.
  • This approach enables the determination of the optimal sites for interaction between chemical entities, including candidate hepatitis C virus polymerase modifiers and hepatitis C virus polymerase. Potential sites for modification within the various binding sites of the molecule can also be identified. This information provides an additional tool for determining the most efficient binding interactions, for example, increased hydrophobic interactions, between hepatitis C virus polymerase and a chemical entity.
  • high resolution x-ray diffraction data collected from crystals exposed to different types of solvent allows the determination of where each type of solvent molecule resides.
  • Small molecules that bind tightly to those sites can then be designed and synthesized and tested for their potential hepatitis C virus polymerase inhibition activity. All of the complexes referred to above may be studied using well-known x-ray diffraction techniques and may be refined versus 1.5-3.5
  • a resolution x-ray data to an R value of about 0.30 or less using computer software, such as X- PLOR (Yale University, 1992, distributed by Molecular Simulations, Inc.; see, e.g., Blundell & Johnson, supra; Meth. Enzymol, Vol. 1 14 & 1 15, H.W.
  • the invention also includes the unique three-dimensional configuration defined by a set of points defined by the structure coordinates for a molecule or molecular complex structurally homologous to hepatitis C virus polymerase as determined using the method of the present invention, structurally equivalent configurations, and magnetic storage media including such set of structure coordinates. Further, the invention includes structurally homologous molecules as identified using the method of the invention.
  • HOMOLOGY MODELING Using homology modeling, a computer model of a hepatitis C virus polymerase homolog can be built or refined without crystallizing the homolog.
  • a preliminary model of the hepatitis C virus polymerase homolog is created by sequence alignment with hepatitis C virus polymerase, secondary structure prediction, the screening of structural libraries, or any combination of those techniques.
  • Computational software may be used to carry out the sequence alignments and the secondary structure predictions.
  • Structural incoherences e.g., structural fragments around insertions and deletions, can be modeled by screening a structural library for peptides of the desired length and with a suitable conformation.
  • a side chain rotamer library may be employed. If the hepatitis C virus polymerase homolog has been crystallized, the final homology model can be used to solve the crystal structure of the homolog by molecular replacement, as described above. Next, the preliminary model is subjected to energy minimization to yield an energy minimized model.
  • the energy minimized model may contain regions where stereochemistry restraints are violated, in which case such regions are remodeled to obtain a final homology model.
  • the homology model is positioned according to the results of molecular replacement, and subjected to further refinement including molecular dynamics calculations.
  • RATIONAL DRUG DESIGN Computational techniques can be used to screen, identify, select and/or design chemical entities capable of associating with hepatitis C virus polymerase or structurally homologous molecules. Knowledge of the structure coordinates for hepatitis C virus polymerase permits the design and/or identification of synthetic compounds and/or other molecules which have a shape complementary to the conformation of the hepatitis C virus polymerase binding site.
  • computational techniques can be used to identify or design chemical entities, such as modifiers, agonists and antagonists, that associate with a hepatitis C virus polymerase binding site or an hepatitis C virus polymerase-like binding site.
  • Potential modifiers may bind to or interfere with all or a portion of a binding site of hepatitis C virus polymerase, and can be competitive, non- competitive, or uncompetitive inhibitors; or interfere with dimerization by binding at the interface between the two monomers. Once identified and screened for biological activity, these inhibitors/agonists/antagonists may be used therapeutically or prophylactically to block hepatitis C virus polymerase activity and, thus, prevent the onset and/or further progression of hepatitis infection. Structure-activity data for analogues of potential modifiers that bind to or interfere with hepatitis C virus polymerase or hepatitis C virus polymerase-like binding sites can also be obtained computationally.
  • chemical entity refers to chemical compounds, complexes of two or more chemical compounds, and fragments of such compounds or complexes.
  • Chemical entities that are determined to associate with hepatitis C virus polymerase are potential drug candidates.
  • Data stored in a machine-readable storage medium that displays a graphical three- dimensional representation of the structure of hepatitis C virus polymerase or a structurally homologous molecule, as identified herein, or portions thereof may thus be advantageously used for drug discovery.
  • the structure coordinates of the chemical entity are used to generate a three-dimensional image that can be computationally fit to the three-dimensional image of hepatitis C virus polymerase or a structurally homologous molecule.
  • the three-dimensional molecular structure encoded by the data in the data storage medium can then be computationally evaluated for its ability to associate with chemical entities.
  • the molecular structures encoded by the data is displayed in a graphical three-dimensional representation on a computer screen, the protein structure can also be visually inspected for potential association with chemical entities.
  • One embodiment of the method of drug design involves evaluating the potential association of a known chemical entity with hepatitis C virus polymerase or a structurally homologous molecule, particularly with a hepatitis C virus polymerase binding site or hepatitis C virus polymerase-like binding site.
  • the method of drug design thus includes computationally evaluating the potential of a selected chemical entity to associate with any of the molecules or molecular complexes set forth above.
  • This method includes the steps of: (a) employing computational means to perform a fitting operation between the selected chemical entity and a binding site or a site nearby the binding site of the molecule or molecular complex; and (b) analyzing the results of said fitting operation to quantify the association between the chemical entity and the binding site.
  • the method of drug design involves computer- assisted design of chemical entities that associate with hepatitis C virus polymerase, its homologs, or portions thereof.
  • Chemical entities can be designed in a step-wise fashion, one fragment at a time, or may be designed as a whole or "de novo."
  • the chemical entity identified or designed according to the method must be capable of structurally associating with at least part of a hepatitis C virus polymerase or hepatitis C virus polymerase-like binding sites, and must be able, sterically and energetically, to assume a conformation that allows it to associate with the hepatitis C virus polymerase or hepatitis C virus polymerase-like binding site.
  • Non-covalent molecular interactions important in this association include hydrogen bonding, van der Waals interactions, hydrophobic interactions, and electrostatic interactions.
  • Conformational considerations include the overall three-dimensional structure and orientation of the chemical entity in relation to the binding site, and the spacing between various functional groups of an entity that directly interact with the hepatitis C virus polymerase-like binding site or homologs thereof.
  • the potential binding of a chemical entity to a hepatitis C virus polymerase or hepatitis C virus polymerase-like binding site is analyzed using computer modeling techniques prior to the actual synthesis and testing of the chemical entity. If these computational experiments suggest insufficient interaction and association between it and the hepatitis C virus polymerase or hepatitis C virus polymerase-like binding site, testing of the entity is obviated.
  • Binding assays to determine if a compound (e.g., an inhibitor) actually interferes with hepatitis C virus polymerase can also be performed and are well known in the art. Binding assays may employ kinetic or thermodynamic methodology using a wide variety of techniques including, but not limited to, microcalorimetry, circular dichroism, capillary zone electrophoresis, nuclear magnetic resonance spectroscopy, fluorescence spectroscopy, and combinations thereof.
  • One method for determining whether a modifier binds to a protein is isothermal denaturation. This method includes taking a sample of a protein (in the presence or absence of substrates) at a fixed elevated temperature where denaturation of the protein occurs in a given time frame, adding the chemical entity to the protein, and monitoring the rate of denaturation. If the chemical entity does bind to the protein, it is expected that the rate of denaturation would be slower in the presence of the chemical entity than in the absence of the chemical entity. For example, this method has been described in Epps et al., Anal. Biochem., 292:40-50 (2001).
  • One skilled in the art may use one of several methods to screen chemical entities or fragments for their ability to associate with a hepatitis C virus polymerase or hepatitis C virus polymerase-like binding site.
  • This process may begin by visual inspection of, for example, a hepatitis C virus polymerase or hepatitis C virus polymerase-like binding site on the computer screen based on the hepatitis C virus polymerase structure coordinates listed in Table 1 or other coordinates which define a similar shape generated from the machine-readable storage medium. Selected fragments or chemical entities may then be positioned in a variety of orientations, or docked, within the binding site.
  • Docking may be accomplished using software such as QUANTA and SYBYL, followed by energy minimization and molecular dynamics with standard molecular mechanics forcefields, such as CHARMM and AMBER. Specialized computer programs may also assist in the process of selecting fragments or chemical entities. Examples include GRID (Goodford, J. Med. Chem., 28:849-57 (1985); available from Oxford University, Oxford, UK);
  • MCSS (Miranker et al., Proteins: Struct. Fund. Gen., 11:29-34 (1991); available from Molecular Simulations, San Diego, CA); AUTODOCK (Goodsell et al., Proteins: Struct. Fund. Genet., 8: 195-202 (1990); available from Scripps Research Institute, La Jolla, CA); and DOCK (Kuntz et al., J. Mol. Biol, 161 :269-88 (1982); available from University of California, San Francisco, CA).
  • 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 of hepatitis C virus polymerase. This would be followed by manual model building using software such as QUANTA or SYBYL (Tripos Associates, St. Louis, MO).
  • Useful programs to aid one of skill in the art in connecting the individual chemical entities or fragments include, without limitation, CAVEAT (P.A. Bartlett et al., in "Molecular Recognition in Chemical and Biological Problems," Special Publ., Royal Chem. Soc, 78: 182-96 (1989); Lauri et al., J. Comput. Aided Mol.
  • Hepatitis C virus polymerase binding compounds may be designed "de novo" using either an empty binding site or optionally including some portion(s) of a known modifier(s). There are many de novo potential modifier design methods including, without limitation, LUDI (Bohm, J. Comp. Aid.
  • an effective hepatitis C virus polymerase or hepatitis C virus polymerase-like binding site modifier must preferably demonstrate a relatively small difference in energy between its bound and free states (i.e., a small deformation energy of binding).
  • the most efficient hepatitis C virus polymerase or hepatitis C virus polymerase-like binding site modifiers should preferably be designed with a deformation energy of binding of at most about 10 kcal/mole; more preferably, at most 7 kcal/mole.
  • Hepatitis C virus polymerase or hepatitis C virus polymerase-like binding site modifiers may interact with the binding site in more than one conformation that is similar in overall binding energy. In those cases, the deformation energy of binding is taken to be the difference between the energy of the free entity and the average energy of the conformations observed when the modifier binds to the protein.
  • An entity designed or selected as binding to or interfering with a hepatitis C virus polymerase or hepatitis C virus polymerase-like binding site may be further computationally optimized so that in its bound state it would preferably lack repulsive electrostatic interaction with the target enzyme and with the surrounding water molecules.
  • Such non-complementary electrostatic interactions include repulsive charge-charge, dipole-dipole, and charge-dipole interactions.
  • Specific computer software is available in the art to evaluate compound deformation energy and electrostatic interactions. Examples of programs designed for such uses include: Gaussian 94, revision C (M.J. Frisch, Gaussian, Inc., Pittsburgh, PA (1995)); AMBER, version 4.1 (P.A. Kollman, University of California at San Francisco, (1995)); QUANT A CHARMM (Molecular Simulations, Inc., San Diego, CA (1995)); Insight II/Discover (Molecular
  • the quality of fit of such entities to the binding site may be judged either by shape complementarity or by estimated interaction energy (Meng et al., J. Comp. Chem., 13:505-24 (1992)).
  • This invention also enables the development of chemical entities that can isomerize to short-lived reaction intermediates in the chemical reaction of a substrate or other compound that interferes with or with hepatitis C virus polymerase. Time-dependent analysis of structural changes in hepatitis C virus polymerase during its interaction with other molecules is carried out. The reaction intermediates of hepatitis C virus polymerase can also be deduced from the reaction product in co-complex with hepatitis C virus polymerase.
  • Such information is useful to design improved analogues of known hepatitis C virus polymerase modifiers or to design novel classes of potential modifiers based on the reaction intermediates of the hepatitis C virus polymerase and modifier co- complex.
  • This provides a novel route for designing hepatitis C virus polymerase modifiers with both high specificity and stability.
  • Yet another approach to rational drug design involves probing the hepatitis C virus polymerase crystal of the invention with molecules including a variety of different functional groups to determine optimal sites for interaction between candidate hepatitis C virus polymerase modifiers and the protein. For example, high resolution x-ray diffraction data collected from crystals soaked in or co-crystallized with other molecules allows the determination of where each type of solvent molecule sticks.
  • Molecules that bind tightly to those sites can then be further modified and synthesized and tested for their hepatitis C virus polymerase modifier activity (Travis, Science, 262:1374 (1993)).
  • iterative drug design is used to identify modifiers of hepatitis C virus polymerase. Iterative drug design is a method for optimizing associations between a protein and a compound by determining and evaluating the three-dimensional structures of successive sets of protein/compound complexes.
  • crystals of a series of protein/compound complexes are obtained and then the three-dimensional structures of each complex is solved. Such an approach provides insight into the association between the proteins and compounds of each complex.
  • a compound that is identified or designed as a result of any of these methods can be obtained (or synthesized) and tested for its biological activity, e.g., inhibition of hepatitis C virus polymerase activity.
  • compositions of this invention include a potential modifier of hepatitis C virus polymerase activity identified according to the invention, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, adjuvant, or vehicle.
  • pharmaceutically acceptable carrier refers to a carrier(s) that is "acceptable” in the sense of being compatible with the other ingredients of a composition and not deleterious to the recipient thereof.
  • the pH of the formulation is adjusted with pharmaceutically acceptable acids, bases, or buffers to enhance the stability of the formulated compound or its delivery form. Methods of making and using such pharmaceutical compositions are also included in the invention.
  • compositions of the invention can be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally, or via an implanted reservoir. Oral administration or administration by injection is preferred.
  • parenteral as used herein includes subcutaneous, intracutaneous, intravenous, intramuscular, intraarticular, intrasynovial, intrasternal, intrathecal, intralesional, and intracranial injection or infusion techniques. Dosage levels of about 0.01 to about 100 mg/kg body weight per day, preferably of about 0.5 to about 75 mg/kg body weight per day of the hepatitis C virus polymerase inhibitory compounds described herein are useful for the prevention and treatment of hepatitis C virus polymerase mediated disease.
  • the pharmaceutical compositions of this invention will be administered about 1 to about 5 times per day or alternatively, as a continuous infusion. Such administration can be used as a chronic or acute therapy.
  • the amount of active ingredient that may be combined with the carrier materials to produce a single dosage form will vary depending upon the host treated and the particular mode of administration.
  • a typical preparation will contain about 5% to about 95% active compound (w/w).
  • such preparations contain about 20% to about 80% active compound.
  • HCV JI NS5BC ⁇ 21 C-terminally truncated for of the HCV polymerase NS5B from the HCV Jl strain was utilized (HCV JI NS5BC ⁇ 21) (Yamashita et al., J. Biol. Chem., 273: 15479-15486 (1998)).
  • a similarly truncated enzyme from another viral strain has been utilized by others in structural studies (Ago et al., Structure, 7: 1417-1426 (1999); Lesburg et al., Nat. Struct. Biol, 6:937-943 (1999); and U.S. Pat. No.
  • the Sf21 cells were harvested by centrifugation at 66 hours post-infection. The cell pellets were snap frozen and stored at -70°C prior to protein purification.
  • the baculovirus infected Sf21 cells were lysed by mechanical means, and the cell lysate was clarified by centrifugation.
  • the clarified cell supernatant was loaded on a Ni NTA column under conditions that allowed binding of the 6-His tag of the recombinant protein.
  • the column was washed free of non-specifically bound cellular proteins and the specifically bound NS5b was eluted with buffer containing imidazole.
  • the column fractions containing NS5b were pooled, concentrated and run over a Superdex 75 gel filtration column. The peak containing NS5b was collected, pooled and the protein concentration was determined by measuring absorbance at 280 nm. Purified protein solution was snap frozen and stored at -70°C.
  • CRYSTALLIZATION PROCEDURE Crystallization began with frozen HCV NS5b protein in buffer containing 20 mM Tris pH 7.5, 20% glycerol, 500 mM NaCl, 10 mM MgCl 2 , 250 mM imidazole, 0.1 mM PMSF, 2 ⁇ g/ml leupeptin, and 5 mM ⁇ ME at a protein concentration of 1.9 mg/ml.
  • the protein was thawed, exchanged into 20 mM HEPES pH 7.5, 20 mM DTT, 1 mM EDTA, 100 mM NaCl, 5% glycerol, and 10 mM MgCl , then concentrated to 20 mg/ml.
  • Raw images obtained from either crystal were integrated and intensities of symmetry equivalent observations scaled with the HKL-2000 program package (Otwinowski, Methods in Enzymology, 276:307-326 (1997)).
  • Structure solution Crystals were assigned to one of the enantiomo ⁇ hic space groups P3-21 or P3 21 based on scaling statistics and systematic absences in the diffraction data aps256.
  • inhibitor PHA-562769 Solid PHA-562769 inhibitor was dissolved in DMSO to make 100 mM stock solution. Inhibitor stock solutions were added to both soaking and cryo-protective solutions to bring the concentration of inhibitor to 2 mM throughout soaking and cryoprotection.
  • the transfers to cryo solutions were done using the same additions as described for the soaking solution, with one added step. Before plunging the crystals in liquid nitrogen, the crystals were dipped in 100% cryo solution for 30 seconds. The total soaking time starts at the beginning of the transfers and also includes the time during cryo solution transfers. The crystals were soaked for 8 hours. Crystals were cryo-cooled in liquid nitrogen and stored. Data Collection.
  • the detector intercepts 2.6 A data.
  • Raw images were integrated and intensities of symmetry equivalent observations scaled with the HKL-2000 program package.
  • the structures of HCVpol/PHA- 562769 complex was phased by molecule replacement using the software as described above, with the native enzyme atomic structure as the search model.
  • the soaking solution with compound was gradually added directly to the drops containing native crystals over approximately 1 hour. Soaking solution was added in the following volumes with approx 10 minutes between additions: O.lul, 0.25ul, 0.5ul, l.Oul, 2.0ul, -2ul +2ul.
  • the transfers to cryo solutions were done using the same additions as described for the soaking solution, with one added step.
  • the crystals were dipped in 100% cryo solution for 30 seconds. The total soaking time starts at the beginning of the transfers and also includes the time during cryo solution transfers. The crystals were soaked for 8 hours. Crystals were cryo-cooled in liquid nitrogen and stored prior to data collection. Data Collection.
  • Structure phasing and refinement The structures of HCVpol/PHA- 729145 complex was phased by molecule replacement using the software as described above, with the PHA-562769 complex model atomic coordinates with inhibitor removed as the search model. Difference electron density (F 0 -F c ) and (2F 0 -F C ) computed with molecular replacement model phases were examined to identify inhibitor binding positions.
  • Complex model atomic coordinates were refined with CNX using the same procedure outlined in Example 1. Final summary statistics are given in Table 14. Residues neighboring the PHA-729145 binding site are listed in Tables 15-17.
  • SEQUENCE LISTING FREE TEXT SEQ ID NO: 1 residues for hepatitis C virus polymerase is from HCV genotype IB isolate Jl (C-delta-21)

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Abstract

La structure cristalline par rayons X de la polymérase du virus de l'hépatite C, ou celle de protéines de type polymérase du virus de l'hépatite C, est utilisée pour dissoudre la structure d'autres molécules ou complexes moléculaires, ainsi que pour identifier et/ou détecter les modificateurs potentiels de l'activité de la polymérase du virus de l'hépatite C.
PCT/US2004/016763 2003-06-05 2004-05-27 Cristaux de la polymerase de l'hepatite c et/ou des proteines de type polymerase du virus de l'hepatite c et utilisation associee WO2005001417A2 (fr)

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Cited By (1)

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WO2013106639A1 (fr) * 2012-01-13 2013-07-18 Gilead Pharmasset Llc Structure cristalline de complexes de polymérase de vhc et procédés d'utilisation

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US6906190B2 (en) * 2002-01-04 2005-06-14 Ribapharm Inc. Inhibitors for de novo-RNA polymerases and methods of identifying targets for same

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013106639A1 (fr) * 2012-01-13 2013-07-18 Gilead Pharmasset Llc Structure cristalline de complexes de polymérase de vhc et procédés d'utilisation

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