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WO2001083717A2 - Conception de modulateurs pour galactosyltransferases - Google Patents

Conception de modulateurs pour galactosyltransferases Download PDF

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Publication number
WO2001083717A2
WO2001083717A2 PCT/CA2001/000607 CA0100607W WO0183717A2 WO 2001083717 A2 WO2001083717 A2 WO 2001083717A2 CA 0100607 W CA0100607 W CA 0100607W WO 0183717 A2 WO0183717 A2 WO 0183717A2
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atom
galactosyltransferase
binding domain
ligand
ligand binding
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WO2001083717A3 (fr
Inventor
Mohan Rao
Igor Tvaroska
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Glycodesign Inc
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Glycodesign Inc
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1048—Glycosyltransferases (2.4)
    • C12N9/1051—Hexosyltransferases (2.4.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y204/00—Glycosyltransferases (2.4)
    • C12Y204/01—Hexosyltransferases (2.4.1)
    • C12Y204/01087—N-Acetyllactosaminide 3-alpha-galactosyltransferase (2.4.1.87), i.e. alpha-1,3-galactosyltransferase
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2299/00—Coordinates from 3D structures of peptides, e.g. proteins or enzymes

Definitions

  • the invention relates to structures and models of ligand binding domains of galactosyltransferases, and the ligand binding domains with ligands.
  • the structural coordinates that define the structures and any ligands bound to the structures enable the determination of homologues, the structures of polypeptides with unknown structure, and the identification of modulators of the galactosyltransferases.
  • the invention also relates ( to structures and models of nucleotide-sugar donors for the galactosyltransferases, and the design of modulators for the galactosyltransferases based on the properties of these structures and models. BACKGROUND OF THE INVENTION .
  • Carbohydrate groups of glycoproteins are involved in various signaling and molecular recognition processes leading to important biological functions (1) and diseases (2).
  • the processing and synthesis of a large number of both N- and O- linked carbohydrate chains involve the sequential and coordinated action of many different glycosyltransferases.
  • Glycosyltransferases catalyze the transfer of monosaccharide from nucleotide sugars to a specific hydroxyl of various saccharide acceptors that leads to the formation of a new glycosidic linkage. There is at least one distinct glycosyltransferase for every type of glycosidic linkage.
  • Galactosyltransferases are a class of enzymes that utilize uridine-5'-diphosphogalactose (UDP-Gal) as the donor. Recently, a retaining galactosyltransferase, ⁇ -l,3-galactosyltransferase ( ⁇ -l-3GalT; E.C.2.4.1.151) (4) has attracted much attention due to a problem of organ rejection in xenotransplantation. This enzyme is responsible for the formation of terminal ⁇ -Gal sequences in Gala 1-3 Gal ⁇ l- GlcNAc ⁇ l-R.
  • Oligosaccharide structures with a terminal Gal ⁇ l-3Gal ⁇ sequence are xenoactive antigens (5) and are considered to be the major cause of hyperacute rejections in xenotransplantation.
  • ⁇ - 1,3 -Galactosyltransferase is absent in humans and, conversely, large quantities of natural anti- ⁇ -l,3-Gal antibodies exist in the human body which react with the ⁇ -Gal epitope, thus providing a barrier to xenotransplant.
  • the appearance of aberrant ⁇ -l,3-GalT in human cells is assumed to be responsible for some forms of anti-immune diseases (6).
  • Galactosyltransferases share a common topology with type II membrane proteins.
  • Type II membrane proteins generally have a large N-terminal catalytic domain, a short stem region and a hydrophobic rich transmembrane domain (3).
  • various groups have performed a host of biochemical studies on this enzyme to understand structure-function relationships, the actual binding and catalytic mechanism of ⁇ -l,3-GalT is poorly understood.
  • it is essential to have a three- dimensional structure of ⁇ -l,3-GalT and structural information about the binding of UDP-Gal and oligosaccharide acceptor in the active site of ⁇ -l,3-GalT.
  • no crystal structure is available on ⁇ -l,3-GalT in native or complexed form.
  • the present inventors have produced a homology model for galactosyltransferases, and complexes of the enzymes with ligands including UDP and UDP-Gal.
  • the homology model was developed by means of molecular modeling using the SpsA glycosyltransferase structure.
  • a protein-ligand docking approach was used to model ⁇ -l,3-GalT complexed with UDP and UDP-Gal.
  • the diphosphate interacts with a DVD motif (Asp-225, Val-226 and Asp-227) of ⁇ -l-3GalT through a Mn 2+ cation.
  • the uridine part of the UDP binds into the cavity that consists of Phe-134, Tyr-139, Ile-140, Val-136, Arg-194, Arg-202, Lys-209, Asp- 173, His-218, and Thr-137, in a "canonical conformation”.
  • Structural features of the ⁇ -l,3-GalT model were compared with available stractural data on this class of enzymes and revealed similarities in the UDP binding pocket.
  • the invention provides a model or secondary, tertiary, and/or quanternary structure of a ligand binding domain of a galactosyltransferase. Binding domains are of significant utility in drug discovery. The association of natural ligands and substrates with the binding domains of galactosyltransferases is the basis of biological mechanisms. The associations may occur with all or any parts of a binding domain. An understanding of these associations will lead to the design and optimization of drugs having more favorable associations with their target enzyme and thus provide improved biological effects.
  • Ligand binding domains include one or more of the binding domains for a disphosphate group of a sugar nucleotide donor, a nucleotide of a sugar nucleotide donor, a nitrogeneous heterocyclic base (preferably a pyrimidine base, more preferably uracil) of a sugar nucleotide donor, a sugar of the nucleotide of a sugar nucleotide donor, a selected sugar of a sugar nucleotide donor that is transferred to an acceptor, and/or an acceptor.
  • the structure of a ligand binding domain may be defined by selected binding sites in the domain.
  • the present invention provides a model or a secondary or three dimensional structure of a ligand binding domain of a galactosyltransferase comprising one or more of the amino acid residues shown in Table 1 or Figure 2, 3, 4, or 6.
  • the mvention also relates to a model or a secondary or three dimensional structure of a ligand binding domain of a galactosyltransferase defined by the structural coordinates of one or more of the atomic interactions or contacts of Table 1.
  • Each of the atomic interactions is defined in Table 1 by an atomic contact (more preferably a specific atom where indicated) on the sugar nucleotide donor and an atomic contact (more preferably a specific atom where indicated) on the galactosyltransferase.
  • a model of a ligand binding domain designed in accordance with a method of the invention and comprising hydrogen binding partners for the amide hydrogen, carbonyl oxygen in position 4, and the carbonyl oxygen of uracil.
  • the invention also provides a model of a ligand binding domain that binds the uridine portion of UDP and comprises two or more of Phe-134, Tyr-139, IIe-140, Val-136, Arg-194, Arg-202, Lys-209 (numbered as ATOM
  • the invention also provides a model of a ligand binding domain that interacts with a pyrophosphate portion of UDP comprising Asp-225, Val-226, and Asp-227.
  • the invention provides a model or secondary, tertiary and/or quanternary structure of a galactosyltransferase.
  • the invention contemplates a model or secondary, tertiary and/or quanternary structure of a galactosyltransferase in association with a ligand or substrate.
  • the structures and models of the invention provide information about the atomic contacts involved in the interaction between the enzyme and a known ligand which can be used to screen for unknown ligands. Therefore the present invention provides a method of screening for a ligand capable of binding a galactosyltransferase ligand binding domain, comprising the use of a secondary or three-dimensional structure or a model of the invention.
  • the method may comprise the step of contacting a ligand binding domain with a test compound, and determining if the test compound binds to the ligand.
  • a method of the invention may identify a ligand which can modulate the biological activity of a galactosyltransferase.
  • a ligand is referred to herein as a "modulator".
  • the present mvention contemplates a method of identifying a modulator of a galactosyltransferase or a ligand binding domain or binding site thereof, comprising the step of using the structural coordinates of a galactosyltransferase or a ligand binding domain or binding site thereof, or a model of the invention to computationally evaluate a test compound for its ability to associate with the galactosyltransferase or ligand binding domain or binding site thereof.
  • Use of the stractural coordinates of a galactosyltransferase stracture, ligand binding domain, or binding site thereof, of the invention to identify a ligand or modulator is also provided.
  • a stracture or model of the invention may be used to design, evaluate, and identify ligands of galactosyltransferases other than ligands that associate with a galactosyltransferase.
  • the ligands may be based on the shape and structure of a galactosyltransferase, or a ligand binding domain or atomic interactions, or atomic contacts thereof. Therefore, ligands, in particular modulators, may be derived from ligand binding domains or analogues or parts thereof.
  • the present invention also contemplates a ligand identified by a method of the invention.
  • a ligand may be a competitive or non-competitive inhibitor of a galactosyltransferase.
  • the ligand is capable of modulating the activity of a galactosyltransferase enzyme.
  • a method for identifying a potential modulator of a galactosyltransferase by determining binding interactions between a test compound and atomic contacts of a binding domain of a galactosyltransferase defined in accordance with the invention comprising:
  • test compounds that are potential modulators by their ability to enter into a selected number of atomic contacts.
  • Another aspect of the invention provides methods for identifying a potential modulator of a galactosyltransferase function by docking a computer representation of a test compound with a computer representation of a structure of a galactosyltransferase or a ligand binding domain thereof that is defined as described herein. In an embodiment the method comprises the following steps:
  • test compounds that best fit the atomic interactions or contacts as potential modulators of the galactosyltransferase.
  • the method comprises the following steps:
  • the ligands or compounds identified according to the methods of the invention preferably have structures such that they are able to enter into an association with a ligand binding domain.
  • Selected ligands or compounds may be characterized by their suitability for binding to particular binding domains.
  • a ligand binding domain or binding site may be regarded as a type of negative template with which the compounds correlate as positives in the manner described herein and thus the compounds are unambiguously defined. Therefore, it is possible to describe the structure of a compound suitable as a modulator of a galactosyltransferase by accurately defining the atomic interactions to which the compound binds to a ligand binding domain and deriving the stracture of the compound from the spacial stracture of the target.
  • the invention contemplates a method for the design of ligands, in particular modulators, for galactosyltransferases based on the three dimensional structure of a sugar nucleotide donor (or part thereof) defined in relation to its spatial association with the three dimensional stracture of the galactosyltransferase or a ligand binding domain thereof.
  • a method for designing potential inhibitors of a galactosyltransferase comprising the step of using the structural coordinates of a sugar nucleotide donor or part thereof, defined in relation to its spatial association with a three dimensional structure or model of a galactosyltransferase or a ligand binding domain thereof, to generate a compound for associating with a ligand binding domain of the galactosyltransferase.
  • the following steps are employed in a particular method of the invention: (a) generating a computer representation of a sugar nucleotide donor, or part thereof, defined in relation to its spatial association with the three dimensional structure of a galactosyltransferase or a ligand binding domain thereof; (b) searching for molecules in a data base that are similar to the defined sugar nucleotide donor, or part thereof, using a searching computer program, or replacing portions of the compound with similar chemical structures from a database using a compound building computer program.
  • the invention further contemplates classes of ligands, in particular modulators, of a galactosyltransferase based on the three-dimensional structure of a sugar nucleotide donor, or part thereof, defined in relation to the sugar nucleotide donor's spatial association with a three dimensional stracture of a galactosyltranferase.
  • a ligand or modulator of a galactosyltransferase may be identified by generating an actual secondary or three-dimensional model of a ligand binding domain or binding site, synthesizing a compound, and examining the components to find whether the required interaction occurs.
  • Modulators which are capable of modulating the activity of galactosyltransferases have therapeutic and prophylactic potential. Therefore, the methods of the invention for identifying modulators may comprise one or more of the following additional steps:
  • Steps (a), (b) (c) and (d) may be carried out in any order, at different points in time, and they need not be sequential.
  • a pharmaceutical composition comprising a modulator, and a method of treating and/or preventing disease comprising the step of administering a modulator or pharmaceutical composition comprising a modulator to a mammalian patient.
  • the invention contemplates a method of treating a disease associated with a galactosyltransferase with inappropriate activity in a cellular organism, comprising:
  • the invention provides for the use of a modulator identified by the methods of the invention in the preparation of a medicament to treat a disease associated with a galactosyltransferase with inappropriate activity in a cellular organism.
  • Use of the stractural coordinates of a galactosyltransferase structure of the invention to manufacture a medicament is also provided.
  • Another aspect of the invention provides machine readable media encoded with data representing a model of the invention or the coordinates of a structure of a galactosyltransferase or ligand binding domain or binding site thereof as defined herein, or the three dimensional structure of a sugar nucleotide donor defined in relation to its spatial association with a three dimensional stracture of a galactosyltransferase as defined herein.
  • the invention also provides computerized representations of a model of the invention or the secondary or three-dimensional structures of the invention , including any electronic, magnetic, or electromagnetic storage forms of the data needed to define the structures such that the data will be computer readable for purposes of display and/or manipulation.
  • the invention further provides a computer programmed with a homology model of a ligand binding domain of a galactosyltransferase.
  • the invention still further contemplates the use of a homology model of the invention as input to a computer programmed for drug design and/or database searching and/or molecular graphic imaging in order to identify new ligands for galactosyltransferases.
  • FIG. 2 A superposition of the SpsA stracture and the ⁇ -l,3-GalT model.
  • the active site residues of SpsA and the corresponding residues of ⁇ -l,3-GalT are shown as tubes.
  • SpsA is shown in magenta and ⁇ -l,3-GalT is in blue.
  • the side-chains of the ⁇ -l,3-GalT model are labeled.
  • the active site modeled metal ion is shown as a red sphere.
  • FIG. 3 The low-energy computed docking modes of UDP to the ⁇ -l,3-GalT. About 60 low energy binding modes of UDP are shown in colored lines. The lowest energy binding mode is shown in thick tube. The critical amino acid residues are shown and labeled. All the low energy binders assume similar binding orientation.
  • FIG. 4 Possible docking modes of UDP-Gal to the ⁇ l,3-GalT.
  • the lowest-energy docking mode is shown as thick tube and some of the low energy binding modes are shown as thin lines.
  • Figure 5 The predicted complex of ⁇ -l-3GalT and the inhibitor. Two top ranking docking modes are shown and in both, the inhibitor occupies the acceptor and pyrophosphate binding regions of the ⁇ -l,3-GalT. The lowest energy-binding mode is shown in thick tube.
  • Figure 6 shows the overall view of a docking model of bovine alpha 1,3 galT-UDP complex.
  • GalT is shown in colored ribbon.
  • the UDP is shown in think tubes.
  • the amino acid residues that interact with UDP are shown in tubes and the modeled Mn 2+ is shown in a sphere.
  • the conserved DVD motif interaction with a metal can be seen.
  • Figure 7 shows an overall representation of the UDP-Gal complex.
  • Figure 8 shows computed low energy binding modes of UDP-Gal.
  • Figure 9 shows lowest energy binding modes of LacNAc- ⁇ -Ome to ⁇ -l,3-GalT. DESCRIPTION OF THE TABLES
  • Table 2 Characterization of the top five binding modes of UDP to ⁇ -l,3-galactosyltransfease.
  • Table 3 Predicted secondary structures for the ⁇ -l,3-GalT sequence that was used for generating a homology model of ⁇ -l,3-GalT.
  • Table 6 Structural coordinates of UDP-Gal.
  • Table 7 Stractural coordinates of uracil, ribose, and pyrophosphate of UDP.
  • the second column identifies the atom number; the third identifies the atom type; the fourth identifies the amino acid type; the fifth identifies the residue number; the sixth identifies the x coordinates; the seventh identifies the y coordinates; and the eighth identifies the z coordinates.
  • association refers to a condition of proximity between a ligand, chemical entity or compound or portions or fragments thereof, and a galactosyltransferase, or portions or fragments thereof (e.g. ligand binding domain).
  • the association may be non-covalent i.e. where the juxtaposition is energetically favored by for example, hydrogen-bonding, van der Waals, or electrostatic or hydrophobic interactions, or it may be covalent.
  • galactosyltransferase refers to an enzyme that catalyzes the transfer of a single monosaccharide unit i.e. galactose, from a donor to the hydroxyl group of an acceptor saccharide.
  • the acceptor can be either a free saccharide, glycoprotein, glycolipid, or polysaccharide.
  • the donor can be a sugar nucleotide, preferably UDP-Gal.
  • Galactosyltransferases show a precise specificity for both the sugar acceptor and donor and generally require the presence of a metal cofactor.
  • Galactosyltransferases are derivable from a variety of sources, including viruses, bacteria, fungi, plants, and animals.
  • the galactosytransferases are derivable from an animal, preferably a mammal including but not limited to bovine, ovine, porcine, murine equine, most preferably a human.
  • the enzyme may be from any source, whether natural, synthetic, semi-synthetic, or recombinant.
  • the galactosyltransferase is a ⁇ l-3 galactosyltransferase, preferably derivable from bovine.
  • a galactosyltransferase or part thereof in the present invention may be a wild type enzyme, or part thereof, or a mutant, variant or homologue of such an enzyme.
  • wild type refers to a polypeptide having a primary amino acid sequence which is identical with the native enzyme (for example, the mammalian enzyme).
  • mutant refers to a polypeptide having a primary amino acid sequence which differs from the wild type sequence by one or more amino acid additions, substitutions or deletions.
  • the mutant has at least 90% sequence identity with the wild type sequence.
  • the mutant has 20 mutations or less over the whole wild-type sequence. More preferably the mutant has 10 mutations or less, most preferably 5 mutations or less over the whole wild-type sequence.
  • a mutant may or may not be functional.
  • variant refers to a naturally occurring polypeptide which differs from a wild-type sequence.
  • a variant may be found within the same species (i.e. if there is more than one isoform of the enzyme) or may be found within a different species.
  • the variant has at least 90% sequence identity with the wild type sequence.
  • the variant has 20 mutations or less over the whole wild-type sequence. More preferably, the variant has
  • polypeptide indicates that the polypeptide comprises a fraction of the wild-type amino acid sequence. It may comprise one or more large contiguous sections of sequence or a plurality of small sections. The "part” may comprise a ligand binding domain as described herein.
  • the polypeptide may also comprise other elements of sequence, for example, it may be a fusion protein with another protein. Preferably the polypeptide comprises at least
  • homologue means a polypeptide having a degree of homology with the wild-type amino acid sequence.
  • homology can be equated with "identity”.
  • a homologous sequence is taken to include an amino acid sequence which may be at least 75, 85 or 90% identical, preferably at least 95 or 98% identical to the wild-type sequence.
  • the homologues will comprise the same sites (for example ligand binding domain) as the subject amino acid sequence.
  • homology can also be considered in terms of similarity (i.e. amino acid residues having similar chemical properties/functions), in the context of the present invention it is preferred to express homology in terms of sequence identity. Homology comparisons can be conducted by eye, or more usually, with the aid of readily available sequence comparison programs. These commercially available computer programs can calculate % homology between two or more sequences (e.g. Wilbur, W.J. and Lipman, D. J. Proc. Natl. Acad. Sci. USA (1983), 80:726- 730).
  • function refers to the ability of a modulator to enhance or inhibit the association between a galactosyltransferase and a compound, or the activity of the galactosyltransferase.
  • Ligand binding domain refers to a region of a molecule or molecular complex that as a result of its shape, favourably associates with a ligand or a part thereof.
  • it may be a region of a galactoysltransferase that is responsible for binding a substrate or known modulator.
  • ligand binding domain includes homologues of a ligand binding domain or portions thereof.
  • the term “homologue” in reference to a ligand binding domain refers to a ligand binding domain or a portion thereof which may have deletions, insertions or substitutions of amino acid residues as long as the binding specificity of the molecule is retained.
  • deliberate amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues as long as the binding specificity of the ligand binding domain is retained.
  • portion thereof means the stractural coordinates corresponding to a sufficient number of amino acid residues of a galactosyltransferase ligand binding domain (or homologues thereof) that are capable of associating with or interacting with a test compound that binds to the ligand binding domain.
  • This term includes galactosyltransferase ligand binding domain amino acid residues having amino acid residues from about 4A to about 5 A of a bound compound or fragment thereof.
  • the structural coordinates provided in the stracture may contain a subset of the amino acid residues in the ligand binding domain which may be useful in the modelling and design of compounds that bind to the ligand binding domain.
  • a ligand binding domain may be defined by its association with a ligand.
  • residues in the ligand binding domain may be defined by their spatial proximity to a ligand.
  • a ligand binding domain of the invention may comprise a DVD motif comprising one or more of Asp-225,
  • a ligand binding domain may comprise one or more of Phe-134, Tyr-139, IIe-140, Val-136,
  • Ligand refers to a compound or entity that associates with a ligand binding domain, including substrates or analogues or parts thereof.
  • a ligand may be designed rationally using a model according to the invention.
  • a ligand may be a modulator.
  • Modulator refers to a molecule which changes or alters the biological activity of a galactosyltransferase.
  • a modulator may increase or decrease galactosyltransferase activity, or change its characteristics, or functional or immunological properties. It may be an inhibitor that decreases the biological or immunological activity of the protein.
  • a modulator may include but is not limited to peptides, members of random peptide libraries and combinatorial chemistry-derived molecular libraries, phosphopeptides (including members of random or partially degenerate, directed phosphopeptide libraries), antibodies, carbohydrates, monosaccharides, oligosaccharides, polysaccharides, glycolipids, saponins, heterocyclic compounds, nucleosides or nucleotides or parts thereof, and small organic or inorganic molecules.
  • a modulator may be an endogenous physiological compound or it may be a natural or synthetic compound.
  • modulator also refers to a chemically modified ligand or compound, and includes isomers and racemic forms.
  • structural coordinates refers to a set of values that define the position of one or more amino acid residues with reference to a system of axes.
  • a data set of stractural coordinates defines the three dimensional structure of a molecule or molecules. Stractural coordinates can be slightly modified and still render nearly identical three dimensional structures.
  • a measure of a unique set of stractural coordinates is the root-mean- square deviation of the resulting stracture. Stractural coordinates that render three dimensional structures that deviate from one another by a root-mean-square deviation of less than 2 A, preferably less than 0.5 A, more preferably less than 0.3 A, may be viewed by a person of ordinary skill in the art as identical.
  • Variations in structural coordinates may be generated because of mathematical manipulations of the structural coordinates of a galactosyltransferase described herein.
  • the stractural coordinates of Table 4 or 8 may be manipulated by crystallographic permutations of the structural coordinates, fractionalization of the stractural coordinates, integer additions or substractions to sets of the stractural coordinates, inversion of the structural coordinates or any combination of the above.
  • Variations in stracture due to mutations, additions, substitutions, and/or deletions of the amino acids, or other changes in any of the components that make up a stracture of the invention may also account for modifications in stractural coordinates. If such modifications are within an acceptable standard error as compared to the original stractural coordinates, the resulting stracture may be the same. Therefore, a ligand that associates with or binds to a ligand binding domain of a galactosyltransferase would also be expected to associate with or bind to another ligand binding domain whose structural coordinates defined a shape that fell within the acceptable error. Such modified structures of a ligand binding domain are also within the scope of the invention.
  • Various computational analyses may be used to determine whether a ligand or the ligand binding domain thereof is sufficiently similar to all or parts of a ligand or ligand binding domain of the invention. Such analyses may be carried out using conventional software applications and methods as described herein.
  • modeling includes the quantitative and qualitative analysis of molecular stracture and/or function based on atomic structural information and interaction models.
  • the term includes conventional numeric- based molecular dynamic and energy minimization models, interactive computer graphic models, modified molecular mechanics models, distance geometry, and other structure-based constraint models.
  • Preferably modeling is performed using a computer and may be optimized using known methods. This is called modeling optimization.
  • substrate refers to molecules that associate with a galactosyltransferase as it catalyzes the transfer of a selected sugar from a nucleotide sugar donor to an acceptor that leads to the formation of a new glycosidic linkage.
  • a substrate includes a sugar nucleotide donor and acceptor and parts thereof.
  • a "sugar nucleotide donor” refers to a nucleotide coupled to a selected sugar that is transferred by a galactosyltransferase to an acceptor.
  • the selected sugar may be a monosaccharide or disaccharide, preferably a monosaccharide.
  • a suitable selected sugar includes galactose.
  • the galatose may be modified for example, the hydroxyls may be blocked with acetonide, acylated, or alkylated or substituted with other groups such as halogen.
  • the nucleotide is preferably UDP.
  • the heterocyclic amine base in the nucleotide may be modified.
  • the base when it is uridine it may be modified at the C-5 or C-6 position with groups including but not limited to alkyl, aryl, gallic acid, and with electron donating and electron withdrawing groups.
  • the sugar in the nucleotide e.g. ribose
  • acceptor refers to the part of a carbohydrate structure (e.g. glycoprotein, glycolipid) where the selected sugar of a sugar nucleotide donor is transferred by the galactosyltransferase.
  • alkyl refers to a branched or linear hydrocarbon radical, typically containing from 1 through 20 carbon atoms, preferably 1 through 10 carbon atoms, more preferably 1 to 6 carbon atoms.
  • Typical alkyl groups include but are not limited to methyl, ethyl, 1 -propyl, 2-propyl, 1 -butyl, 2-butyl, tert- butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, and the like.
  • alkenyl refers to an unsaturated branched or linear group typically having from 2 to 20 carbon atoms and at least one double bond. Examples of such groups include but are not limited to ethenyl, 1-propenyl, 2-propenyl, 1-butenyl, 1,3-butadienyl, 1-hexenyl, 2-hexenyl, 1-pentenyl, 2-pentenyl, and the like.
  • alkynyl refers to an unsaturated branched or linear group having 2 to 20 carbon atoms and at least one triple bond. Examples of such groups include but are not limited to ethynyl, 1- propynyl, 2-propynyl, 1-butynyl, 2-butynyl, 1-pentynyl, and the like.
  • cycloalkyl refers to cyclic hydrocarbon groups and includes but is not limited to cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl.
  • aryl refers to a monocyclic or polycyclic group, preferably a monocyclic or bicyclic group.
  • An aryl group may optionally be substituted as described herein. Examples of aryl groups and substituted aryl groups are phenyl, benzyl, p-nitrobenzyl, p-methoxybenzyl, biphenyl, and naphthyl.
  • alkoxy alone or in combination, refers to an alkyl or cycloalkyl linked to the parent molecular moiety through an oxygen atom.
  • aryloxy refers to an aryl linked to the parent molecular moiety through an oxygen atom.
  • alkoxy groups are methoxy, ethoxy, propoxy, vinyloxy, allyloxy, butoxy, pentoxy, hexoxy, cyclopentoxy, and cyclohexoxy.
  • aryloxy groups are phenyloxy, O-benzyl i.e. benzyloxy, O-p- nitrobenzyl and O-p-methyl-benzyl, 4-nitrophenyloxy, 4-chlorophenyloxy, and the like.
  • halo or halogen, alone or in combination, means fluoro, chloro, bromo, or iodo.
  • amino alone or in combination, refers to a chemical functional group where a nitrogen atom (N) is bonded to three substituents being any combination of hydrogen, alkyl, cycloalkyl, alkenyl, alkynyl, or aryl with the general chemical formula -NR ⁇ 4 R ⁇ 6 where R M and R 16 can be any combination of hydrogen, alkyl, cycloalkyl, alkenyl, alkynyl, or aryl.
  • one substituent on the nitrogen atom can be a hydroxyl group (-OH) to give an amine known as a hydroxylamine.
  • amino groups are amino (-NH 2 ), methylamine, ethylamine, dimethylamine, 2-propylamine, butylamine, isobutylamine, cyclopropylamine, benzylamine, allylamine, hydroxylamine, cyclohexylamino (-NHCH(CH 2 ) 5 ), piperidine (-N(CH 2 ) 5 ) and benzylamino (-NHCH 2 C 6 H 5 ).
  • thioalkyl refers to a chemical functional group where a sulfur atom (S) is bonded to an alkyl.
  • S sulfur atom
  • Examples of thioalkyl groups are thiomethyl, thioethyl, and thiopropyl.
  • thioaryl refers to a chemical functional group where a sulfur atom (S) is bonded to an aryl group with the general chemical formula -SR 16 where Rj 6 is an aryl group which may be substituted.
  • Examples of thioaryl groups and substituted thioaryl groups are thiophenyl, para-chlorothiophenyl, thiobenzyl, 4-methoxy-thiophenyl, 4-nitro-thiophenyl, and para-nitrothiobenzyl.
  • Heterocyclic rings are molecular rings where one or more carbon atoms have been replaced by hetero atoms (atoms not being carbon) such as for example, oxygen (O), nitrogen (N) or sulfur (S), or combinations thereof.
  • heterocyclic rings include ethylene oxide, tetrahydrofuran, thiophene, piperidine (piperidinyl group), pyridine (pyridinyl group), and caprolactam.
  • a carbocyclic or heterocyclic group may be optionally substituted at carbon or nitrogen atoms with for example, alkyl, phenyl, benzyl or thienyl, or a carbon atom in the heterocyclic group together with an oxygen atom may form a carbonyl group, or a heterocyclic group may be fused with a phenyl group.
  • the present invention provides a galactosyltransferase secondary, tertiary and/or quanternary structure.
  • the invention also provides a homology model that represents the secondary, tertiary, and/or quanternary stracture of a galactosyltransferase.
  • a model may, for example, be a structural model (or representation thereof), or a computer model.
  • the model itself may be in two or three dimensions. It is possible for a computer model to be in three dimensions despite the constraints imposed by a conventional computer screen, if it is possible to scroll along at least a pair of axes, causing "rotation" of the image.
  • a method for designing a homology model of a ligand binding domain of a galactosytransferase wherein the homology model may be displayed as a three- dimensional image comprising: (i) providing an amino acid sequence and structural coordinates of a ligand binding domain structure of a glycosyltransferase, preferably SpsA glycosytransferase; (ii) modifying said structure to take into account differences between the amino acid configuration of the ligand binding domains of the galactosyltransferase on the one hand and the SpsA glycosyltransferase on the other hand to generate a homology model, and (iii) if required refining the homology model.
  • the method may further comprise comparing the homology model with the structures of other, similar, proteins.
  • a model or structure of a preferred galactosyltransferase of the invention has the atomic stractural coordinates as shown in Table 4 or Table 8.
  • Computer representations of the stracture i.e. models are illustrated in the Figures.
  • the structural coordinates in a structure or model of the invention may comprise the amino acid residues of a galactosyltransferase ligand binding domain, or a portion or homolog thereof useful in the modeling and design of test compounds capable of binding to the galactosyltransferase. Therefore, the invention also relates to a secondary and three dimensional structure or model of a ligand binding domain of a galactosyltransferase.
  • Ligand binding domains include the ligand binding domains for a disphosphate group of a sugar nucleotide donor, a nucleotide of a sugar nucleotide donor, a nitrogeneous heterocyclic base (preferably a pyrimidine base, more preferably uracil) of a sugar nucleotide donor, and/or a sugar (e.g. galactose) of a sugar nucleotide donor.
  • the structure of a ligand binding domain may be defined by selected atomic interactions or contacts in the domain, preferably two or more of the atomic interactions or contacts as defined in Table 1.
  • a stracture or model of the invention includes a structure where at least one amino acid residue is replaced with a different amino acid residue or by adding or deleting amino acid residues, and having substantially the same three dimensional structure as the galactosyltransferase as described in Table 4 and the Figures, or the ligand binding domains as described in Table 1 (and further defined by the structural coordinates of the ATOMS in Table 4 or Table 8), i.e.
  • a set of atomic structural coordinates that have a root mean square deviation of less than or equal to about 2A, preferably less than 0.5A, most preferably less than 0.3A, when superimposed with the atomic structure coordinates of the galactosyltransferase as described in Table 4 or Table 8, or the binding domains as described in Table 1 (and further defined by the stractural coordinates of the ATOMS in Table 4) when at least 50% to 100% of the atoms of the sugar nucleotide donor binding domain or binding domains of components of the donor as the case may be, are included in the superimposition.
  • the invention also features a secondary and three dimensional structure or model of a galactosyltransferase in association with one or more molecules (e.g. substrates such as UDP-Gal, uridine-ribose, monophophate-Mn 2+ , or diphosphate-Mn 2+ ).
  • the association may be covalent or non-covalent.
  • the molecule may be any organic molecule, and it may modulate the function of a galactosyltransferase by for example inhibiting or enhancing its function, or it may be an acceptor or donor for the galactosyltransferase. It is preferred that the geometry of the compound and the interactions formed between the compound and the galacytosyltransferase provide high affinity binding between the two molecules.
  • the stracture and model of the galactosyltransferase decribed herein has allowed the identification and characterization of the binding domain of UDP and UDP-Gal.
  • the UDP-Gal binding domain has been subdivided into three sub-sites (the uracil-binding domain, the ribose-binding domain, the diphosphate-Mn 2+ binding domain, and the Gal binding domain) and characterized.
  • a secondary and three dimensional structure or model of a ligand binding domain of a galactosyltransferase that binds a diphosphate of a sugar nucleotide donor comprising at least two of atomic interactions 9, 10, and 11 of Table 1, each atomic interaction defined therein by an atomic contact (more preferably, a specific atom where indicated) on the diphosphate, and an atomic contact (more preferably, a specific amino acid residue where indicated) on the galactosyltransferase (i.e. enzyme atomic contact).
  • the ligand binding domain comprises atomic interactions 9 and 10, 10 and 11, 9 and 11, or 9, 10, and 11 of Table 1.
  • the binding domain is defined by the atoms of the enzyme atomic interactions having the stractural coordinates for the atoms listed in Table 4 or Table 8. Therefore, in an embodiment of the invention the binding domain is defined by the stractural coordinates referred to as ATOM 1690, and ATOM 1718 of Table 8most preferably ATOM 1690 to ATOM 1718 inclusive of Table 8.
  • the binding domain of a galactosyltransferase for a diphosphate of a sugar nucleotide donor is also characterized by a DVD motif (Asp-225, Val-226, and Asp-227).
  • a secondary or three dimensional stracture or model of a ligand binding domain of a galactosyltransferase that binds a heterocyclic amine base of a sugar nucleotide donor comprising at least two, preferably three, of atomic interactions 1, 2, 3, and 4 of Table 1, each atomic interaction defined therein by an atomic contact (more preferably, a specific atom where indicated) on the heterocyclic amine base, and an atomic contact (more preferably, a specific amino acid residue where indicated) on the galactosyltransferase (i.e. enzyme atomic contact).
  • the ligand binding domain comprises atomic interactions 1 and 2; 1 and 3; 1 and 4; 2 and 3; 2 and 4; 3 and 4; or 1, 2, and 3; 2, 3, and 4; 1, 3, and 4; 1, 2, and 4; or 1, 2, 3 and 4 of Table 1.
  • the binding domain is defined by the atoms of the enzyme atomic interactions having the stractural coordinates for the atoms listed in Table 4 or Table 8. Therefore, in an embodiment of the invention the binding domain is defined by the structural coordinates referred to as ATOM 720, ATOM 1360, ATOM 1490, ATOM 154 to ATOM 155 in Table 8.
  • the ligand binding domain of a galactosyltransferase for a heterocyclic amine base of a sugar nucleotide donor is also characterized by two helices and two ⁇ sheets in anti-parallel fashion.
  • a ligand binding domain for uracil can also be characterized by the following three hydrogen bonds: (1) the amide hydrogen of uracil in position 3 and OD1 of Asp-168, (2) the carbonyl oxygen of uracil in position 4 and the side chain of Lys-204, and (3) the carbonyl oxygen of uracil in position 2 and the amide hydrogen of the His-213 side chain.
  • a secondary and three dimensional structure or model of a ligand binding domain of a galactosyltransferase that binds the sugar of the nucleotide (e.g. ribose) of a sugar nucleotide donor comprising at least two, preferably three, of atomic interactions 5, 6, 7, and 8 of Table 1, each atomic interaction defined therein by an atomic contact (more preferably, a specific atom where indicated) on the sugar, and an atomic contact (more preferably, a specific amino acid residue where indicated) on the galactosyltransferase (i.e. enzyme atomic contact).
  • the binding domain comprises atomic interactions 5 and 6; 5 and 7; 5 and 8; 6 and 7; 6 and 8; 7 and 8; 5, 6, and 7; 5, 6, and 8; 6, 7, and 8; 5, 7, and 8; and 5, 6, 7,and 8 of Table 1.
  • the ligand binding domain is defined by the atoms of the enzyme atomic interactions having the structural coordinates for the atoms listed in Table 4 or Table 8. Therefore, in an embodiment of the invention the binding domain is defined by the structural coordinates referred to as ATOM 1690, ATOM 97 to ATOM 115, ATOM 1436 to ATOM 1454 of Table 8.
  • Atomic interactions 1 through 11 in Table 1 are preferably each characterized by the types of binding and/or the distances between atomic contacts indicated in Table 1.
  • a secondary or three dimensional structure of a ligand binding domain of a galactosyltransferase that binds a nucleotide (preferably UDP) of a sugar nucleotide donor comprising at least two or more of atomic interactions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 of Table 1, each atomic interaction defined therein by an atomic contact (more preferably, a specific atom where indicated) on the nucleotide, and an atomic contact (more preferably, a specific amino acid residue where indicated) on the galactosyltransferase (i.e. enzyme atomic contact).
  • the binding domain comprises atomic interactions 2, 3, 5, 6, , 9, 10, and 11; 4, 7, 8, 9, 10, and 11; 1, 2, 3, 5, 6, 9, 10, 11, or 1 to 11 inclusive of Table 1.
  • the ligand binding domain is defined by the atoms of the enzyme atomic interactions having the stractural coordinates for the atoms listed in Table 4 or Table 8. Therefore, in an embodiment of the invention the ligand binding domain is defined by the structural coordinates referred to as ATOM 720, ATOM 1360, ATOM 1490, ATOM 154, ATOM 155, ATOM 1690, ATOM 97 to ATOM 115, ATOM 1436 to ATOM 1454, and ATOM 1718, of Table 8.
  • the binding domain of a galactosyltransferase for a nucleotide of a sugar nucleotide donor is also characterized by a 100 amino acid nucleotide recognition domain.
  • a UDP binding domain of a galactosyltransferase is also characterized by an open ⁇ , ⁇ -sandwich made up of three helices packed against four ⁇ -sheets.
  • the following amino acid residues have also been identified to be part of the UDP binding domain: Phe-134, Typ-139, IIe-140, Val-136, Arg-194, Arg-202, Lys-209, Asp-173, His-218, Thr-137, Asp-225, Val-226, and Asp-227.
  • a secondary and three dimensional structure or model of a ligand binding domain of a galactosyltransferase that binds a sugar nucleotide donor comprising at least three of the atomic interactions of Table 1, each atomic interaction defined therein by an atomic contact (more preferably, a specific atom where indicated) on the sugar nucleotide donor, and an atomic contact (more preferably, a specific amino acid residue where indicated) on the galactosyltransferase (i.e. enzyme atomic contact).
  • the binding domain comprises atomic interactions 1 to 11 inclusive of Table 1.
  • the ligand binding domain is defined by the atoms of the enzyme atomic interactions having the stractural coordinates for the atoms listed in Table 4 or Table 8. Therefore, in an embodiment of the invention the ligand binding domain is defined by the structural coordinates referred to as ATOM 720, ATOM 1360, ATOM 1490, ATOM 154, ATOM 155, ATOM 1690, ATOM 97 to ATOM 115, ATOM 1436 to ATOM 1454, and ATOM 1718 of Table 4. Identification of Homologues
  • Information derivable from the structures of the present invention for example the structural coordinates
  • a model of the present invention may be provided in a computer-readable format.
  • the invention provides a computer readable medium or a machine readable storage medium which comprises the models of the invention or structural coordinates of a galactosyltransferase including all or any parts of the galactosyltransfersae (e.g ligand-binding domain), ligands including portions thereof, or substrates including portions thereof.
  • Such storage medium or storage medium encoded with these data are capable of displaying on a computer screen or similar viewing device, a three-dimensional graphical representation of a molecule or molecular complex which comprises the enzyme or ligand binding domains or similarly shaped homologous enzymes or ligand binding domains.
  • the invention also provides computerized representations of a model or structure of the invention, including any electronic, magnetic, or electromagnetic storage forms of the data needed to define the structures such that the data will be computer readable for purposes of display and/or manipulation.
  • the invention provides a computer for producing a model or three-dimensional representation of a molecule or molecular complex, wherein said molecule or molecular complex comprises a galactosyltransferase or ligand binding domain thereof defined by structural coordinates of galactosyltransferase amino acids or a ligand binding domain thereof, or comprises structural coordinates of atoms of a ligand or substrate, or a three-dimensional representation of a homologue of said molecule or molecular complex, wherein said computer comprises:
  • a machine-readable data storage medium comprising a data storage material encoded with machine readable data wherein said data comprises the structural coordinates of a galactosyltransferase amino acids according to Table 4 or Table 8 or a ligand binding domain thereof, or a ligand according to Table 5, 6, or 7;
  • a homologue may comprise a galactosyltransferase or ligand binding domain thereof, or ligand or substrate that has a root mean square deviation from the backbone atoms of not more than 1.5 angstroms.
  • the invention also provides a computer for determining at least a portion of the structural coordinates corresponding to an X-ray diffraction pattern of a molecule or molecular complex wherein said computer comprises:
  • a machine-readable data storage medium comprising a data storage material encoded with machine readable data wherein said data comprises the structural coordinates according to Table 4, 5, 6, 7, or 8;
  • a machine-readable data storage medium comprising a data storage material encoded with machine readable data wherein said data comprises an X-ray diffraction pattern of said molecule or molecular complex
  • a central-processing unit coupled to said working memory and to said machine-readable data storage medium of (a) and (b) for performing a Fourier transform of the machine readable data of (a) and for processing said machine readable data of (b) into stractural coordinates;
  • a display coupled to said central-processing unit for displaying said stractural coordinates of said molecule or molecular complex.
  • the invention also contemplates a computer programmed with a homology model of a ligand binding domain according to the invention; a machine-readable data-storage medium on which has been stored in machine- readable form a homology model of a ligand binding domain of a galactosyltransferase; and the use of a homology model as input to a computer programmed for drag design and/or database searching and/or molecular graphic imaging in order to identify new ligands for galactosyltransferases.
  • the present invention also provides a method for determining the secondary and/or tertiary structures of a polypeptide by using a model according to the invention.
  • the polypeptide may be any polypeptide for which the secondary and or tertiary structure is uncharacterised or incompletely characterised.
  • the polypeptide shares (or is predicted to share) some stractural or functional homology to a galactosyltransferase, preferably a ⁇ l,3 galactosyltranferase.
  • the polypeptide may show a degree of structural homology over some or all parts of the primary amino acid sequence.
  • the polypeptide may have one or more domains which show homology with a galactosyltransferase domain (Kapitonov and Yu (1999) Glycobiology 9(10): 961-978).
  • the polypeptide may be a galactosyltransferase with a different specificity for a ligand or substrate.
  • the polypeptide may be a galactosyltransferase which requires a different metal cofactor.
  • the polypeptide may be a galactosyltransferase from a different species.
  • the polypeptide may be a mutant of the wild-type galactosyltransferase.
  • a mutant may arise naturally, or may be made artificially (for example using molecular biology techniques).
  • the mutant may also not be "made” at all in the conventional sense, but merely tested theoretically using the model of the present invention.
  • a mutant may or may not be functional.
  • the effect of a particular mutation on the overall two and/or three dimensional structure of a galactosyltransferase and/or the interaction between the enzyme and a ligand or substrate can be investigated.
  • the polypeptide may perform an analogous function or be suspected to show a similar catalytic mechanism to the galactosyltransferase enzyme.
  • the polypeptide may transfer a sugar residue from a sugar nucleotide donor.
  • the polypeptide may also be the same as the polypeptide described herein, but in association with a different ligand (for example, modulator or inhibitor) or cofactor. In this way it is possible to investigate the effect of altering a ligand or compound with which the polypeptide is associated on the structure of a ligand binding domain.
  • Secondary or tertiary stracture may be determined by applying the stractural coordinates of the model of the present invention to other data such as an amino acid sequence, X-ray crystallographic diffraction data, or nuclear magnetic resonance (NMR) data. Homology modeling, molecular replacement, and nuclear magnetic resonance methods using these other data sets are described below.
  • Homology modeling also known as comparative modeling or knowledge-based modeling
  • methods develop a three dimensional model from a polypeptide sequence based on the structures of known proteins (e.g. native or mutated galactosyltransferases).
  • the method utilizes a computer representation of the structure of a galactosyltransferase, or a binding domain or complex of same as described herein, a computer representation of the amino acid sequence of a polypeptide with an unknown structure (additional native or mutated galactosyltransferases), and standard computer representations of the structures of amino acids.
  • the method in particular comprises the steps of; (a) identifying structurally conserved and variable regions in the known stracture; (b) aligning the amino acid sequences of the known stracture and unknown stracture (c) generating coordinates of main chain atoms and side chain atoms in structurally conserved and variable regions of the unknown stracture based on the coordinates of the known structure thereby obtaining a homology model; and (d) refining the homology model to obtain a three dimensional stracture for the unknown structure.
  • This method is well known to those skilled in the art (Greer, 1985, Science 228, 1055; Bundell et al 1988, Eur. J. Biochem.
  • step (a) of the homology modeling method a known galactosyltransferase stracture is examined to identify the structurally conserved regions (SCRs) from which an average structure, or framework, can be constructed for these regions of the protein.
  • SCRs structurally conserved regions
  • VRs Variable regions
  • SCRs generally correspond to the elements of secondary structure, such as alpha-helices and beta-sheets, and to ligand- and substrate-binding sites (e.g. acceptor and donor binding sites).
  • the VRs usually lie on the surface of the proteins and form the loops where the main chain turns.
  • Sequence alignments generally are based on the dynamic programming algorithm of Needleman and Wunsch [J. Mol. Biol. 48: 442-453, 1970]. Current methods include FASTA, Smith-Waterman, and BLASTP, with the BLASTP method differing from the other two in not allowing gaps. Scoring of alignments typically involves construction of a 20x20 matrix in which identical amino acids and those of similar character (i.e., conservative substitutions) may be scored higher than those of different character. Substitution schemes which may be used to score alignments include the scoring matrices PAM (Dayhoff et al., Meth. Enzymol.
  • Alignment based solely on sequence may be used, though other stractural features also may be taken into account.
  • multiple sequence alignment algorithms are available that may be used when aligning a sequence of the unknown with the known structures.
  • Four scoring systems i.e. sequence homology, secondary stracture homology, residue accessibility homology, CA-CA distance homology
  • sequence homology i.e. sequence homology, secondary stracture homology, residue accessibility homology, CA-CA distance homology
  • main chain atoms and side chain atoms both in SCRs and VRs need to be modeled.
  • a variety of approaches may be used to assign coordinates to the unknown.
  • the coordinates of the main chain atoms of SCRs will be transferred to the unknown stracture.
  • VRs correspond most often to the loops on the surface of the polypeptide and if a loop in the known stracture is a good model for the unknown, then the main chain coordinates of the known structure may be copied.
  • Side chain coordinates of SCRs and VRs are copied if the residue type in the unknown is identical to or very similar to that in the known stracture.
  • a side chain rotamer library may be used to define the side chain coordinates.
  • fragment databases may be searched for loops in other proteins that may provide a suitable model for the unknown. If desired, the loop may then be subjected to conformational searching to identify low energy conformers if desired.
  • the structural coordinates of a galactosyltransferase structure may be applied to nuclear magnetic resonance (NMR) data to determine the three dimensional stractures of polypeptides in solution (e.g. additional native or mutated galactosyltransferases).
  • NMR nuclear magnetic resonance
  • the structural coordinates of a polypeptide can guide the NMR spectroscopist to an understanding of the spatical interactions between secondary stractural elements in a polypeptide of related stracture.
  • Information on spatial interactions between secondary structural elements can greatly simplify Nuclear Overhauser Effect (NOE) data from two-dimensional NMR experiments.
  • NOE Nuclear Overhauser Effect
  • applying the structural coordinates after the determination of secondary stracture by NMR techniques simplifies the assignment of NOE's relating to particular amino acids in the polypeptide sequence and does not greatly bias the NMR analysis of polypeptide structure.
  • the invention relates to a method of determining three dimensional stractures of polypeptides with unknown stractures, preferably a native or mutated galactosyltransferases, by applying the structural coordinates of a galactosyltransferase stracture, or ligand binding domain or complex thereof described herein to nuclear magnetic resonance (NMR) data of the unknown structure.
  • NMR nuclear magnetic resonance
  • This method comprises the steps of: (a) determining the secondary structure of an unknown structure using NMR data; and (b) simplifying the assignment of through-space interactions of amino acids.
  • through-space interactions defines the orientation of the secondary structural elements in the three dimensional stracture and the distances between amino acids from different portions of the amino acid sequence.
  • the term "assignment” defines a method of analyzing NMR data and identifying which amino acids give rise to signals in the NMR spectrum. Screening Method
  • the present invention provides a method of screening for a ligand that associates with a ligand binding domain and/or modulates the function of a galactosyltranssferase, by using a structure or a model according to the present invention.
  • the method may involve investigating whether a test compound is capable of associating with or binding a ligand binding domain.
  • a method is provided for screening for a ligand capable of associating with or binding to a ligand binding domain, wherein said method comprises the use of a stracture or model according to the invention.
  • the invention in another aspect, relates to a method of screening for a ligand capable of associating with or binding to a ligand binding domain, wherein the ligand binding domain is defined by the amino acid residue stractural coordinates given herein, the method comprising contacting the ligand binding domain with a test compound and determining if said test compound binds to said ligand binding domain.
  • the present invention provides a method of screening for a test compound capable of interacting with a key amino acid residue of a ligand binding domain of a galactosyltransferase.
  • Another aspect of the invention provides a process comprising the steps of: (a) perfo ⁇ ning the method of screening for a ligand as described above;
  • a further aspect of the invention provides a process comprising the steps of: (a) performing the method of screening for a ligand as described above; (b) identifying one or more ligands capable of binding to a ligand binding domain; and
  • test compound capable of interacting with a key amino acid residue in a galactosyltransferase ligand binding domain further steps may be carried out either to select and/or to modify compounds and/or to modify existing compounds, to modulate the interaction with the key amino acid residues in the galactosyltransferase ligand binding domain.
  • Yet another aspect of the invention provides a process comprising the steps of:
  • test compound means any compound which is potentially capable of associating with a ligand binding domain. If, after testing, it is determined that the test compound does associate with or bind to the ligand binding domain, it is known as a "ligand”.
  • a “test compound” includes, but is not limited to, a compound which may be obtainable from or produced by any suitable source, whether natural or not. The test compound may be designed or obtained from a library of compounds which may comprise peptides, as well as other compounds, such as small organic molecules and particularly new lead compounds.
  • the test compound may be a natural substance, a biological macromolecule, or an extract made from biological materials such as bacteria, fungi, or animal (particularly mammalian) cells or tissues, an organic or an inorganic molecule, a synthetic test compound, a semi-synthetic test compound, a carbohydrate, a monosaccharide, an oligosaccharide or polysaccharide, a glycolipid, a glycopeptide, a saponin, a heterocyclic compound, a structural or functional mimetic, a peptide, a peptidomimetic, a derivatised test compound, a peptide cleaved from a whole protein, or a peptide synthesised synthetically (such as, by way of example, either using a peptide synthesizer or by recombinant techniques or combinations thereof), a recombinant test compound, a natural or a non-natural test compound, a fusion protein or equivalent thereof and mutants
  • the test compound may be screened as part of a library or a data base of molecules.
  • Data bases which may be used include ACD (Molecular Designs Limited), NCI (National Cancer Institute), CCDC (Cambridge Crystallographic Data Center), CAST (Chemical Abstract Service), Derwent (Derwent Information Limited), Maybridge (Maybridge Chemical Company Ltd), Aldrich (Aldrich Chemical Company), DOCK (University of California in San Francisco), and the Directory of Natural Products (Chapman & Hall).
  • Computer programs such as CONCORD (Tripos Associates) or DB -Converter (Molecular Simulations Limited) can be used to convert a data set represented in two dimensions to one represented in three dimensions.
  • Test compounds may be tested for their capacity to fit spatially into a galactosyltransferase ligand binding domain.
  • fit spatially means that the three-dimensional stracture of the test compound is accommodated geometrically in a galactosyltransferase ligand binding domain.
  • the test compound can then be considered to be a ligand.
  • a favourable geometric fit occurs when the surface area of the test compound is in close proximity with the surface area of the cavity or pocket without forming unfavorable interactions or associations.
  • a favourable complementary interaction occurs where the test compound interacts by hydrophobic, aromatic, ionic, dipolar, or hydrogen donating and accepting forces. Unfavourable interactions or associations may be steric hindrance between atoms in the test compound and atoms in the binding site.
  • a method for identifying potential modulators of galactosyltransferase function.
  • the method utilizes the structural coordinates or model of a galactosyltransferase three dimensional structure, or binding domain thereof.
  • the method comprises the steps of (a) docking a computer representation of a test compound from a computer data base with a computer model of a ligand binding domain of a galactosyltransferase; (b) determining a conformation of a complex between the test compound and binding domain with a favourable geometric fit or favorable complementary interactions; and (c) identifying test compounds that best fit the galactosyltransferase binding domain as potential modulators of galactosyltransferase function.
  • the initial galactosyltransferase stracture may or may not have substrates bound to it.
  • a favourable complementary interaction occurs where a compound in a compound-galactosyltransferase complex interacts by hydrophobic, ionic, or hydrogen donating and accepting forces, with the active-site or ligand binding domain of a galactosyltransferase without forming unfavorable interactions.
  • a model of the present invention is a computer model
  • the test compounds may be positioned in a ligand binding domain through computational docking.
  • the model of the present invention is a structural model
  • the test compounds may be positioned in the ligand binding domain by, for example, manual docking.
  • docking refers to a process of placing a compound in close proximity with a galactosyltransferase ligand binding domain, or a process of finding low energy conformations of a test compound/ galactosyltransferase complex.
  • a screening method of the present invention may comprise the following steps:
  • a method which comprises the following steps: (a) docking a computer representation of a test compound from a computer data base with a computer representation of a selected site (e.g. an inhibitor binding domain) on a galactosyltransferase structure or model defined in accordance with the invention to obtain a complex; (b) determining a conformation of the complex with a favourable geometric fit and favourable complementary interactions; and (c) identifying test compounds that best fit the selected site as potential modulators of the galactosyltransferase.
  • a method of the invention may be applied to a plurality of test compounds, to identify those that best fit the selected site.
  • the model used in the screening method may comprise a galactosyltransferase or ligand binding domain thereof either alone or in association with one or more ligands and/or cofactors.
  • the model may comprise the ligand-binding domain in association with a ligand, substrate, or analogue thereof.
  • the selected site under investigation may be the ligand binding domain itself.
  • the test compound may, for example, mimic a known substrate for the enzyme in order to interact with the ligand binding domain.
  • the selected site may alternatively be another site on the enzyme.
  • the selected site may be the ligand binding domain or a site made up of the ligand binding domain and the complexed ligand, or a site on the ligand itself.
  • the test compound may be investigated for its capacity to modulate the interaction with the associated molecule.
  • test compound (or plurality of test compounds) may be selected on the basis of its similarity to a known ligand for the galactosyltransferase.
  • the screening method may comprise the following steps:
  • Searching may be carried out using a database of computer representations of potential compounds, using methods known in the art.
  • the present invention also provides a method for designing ligands for a galactosyltransferase. It is well known in the art to use a screening method as described above to identify a test compound with promising' fit, but then to use this test compound as a starting point to design a ligand with improved fit to the model. A known modulator can also be modified to enhance its fit with a model of the invention. Such techniques are known as "structure-based ligand design" (See Kuntz et al., 1994, Ace. Chem. Res. 27:117; Guida, 1994, Current Opinion in
  • the method may comprise the following steps:
  • mapping chemical features of a test compound such as by hydrogen bond donors or acceptors, hydrophobic/lipophilic sites, positively ionizable sites, or negatively ionizable sites; and (b) adding geometric constraints to selected mapped features.
  • the fit of the modified test compound may then be evaluated using the same criteria.
  • the chemical modification of a group may either enhance or reduce hydrogen bonding interaction, charge interaction, hydrophobic interaction, Van Der Waals interaction or dipole interaction between the test compound and the key amino acid residue(s) of the selected site.
  • the group modifications involve the addition, removal, or replacement of substituents onto the test compound such that the substituents are positioned to collide or to bind preferentially with one or more amino acid residues that correspond to the key amino acid residues of the selected site.
  • Identified groups in a test compound may be substituted with, for example, alkyl, alkoxy, hydroxyl, aryl, cycloalkyl, alkenyl, alkynyl, thiol, thioalkyl, thioaryl, amino, or halo groups.
  • initial substitutions are conservative, i.e., the replacement group will have approximately the same size, shape, hydrophobicity and charge as the original group. It should, of course, be understood that components known in the art to alter conformation should be avoided.
  • a modified test compound model may bind to the selected site and be considered to be a "ligand".
  • Rational modification of groups may be made with the aid of libraries of molecular fragments which may be screened for their capacity to fit into the available space and to interact with the appropriate atoms. Databases of computer representations of libraries of chemical groups are available commercially, for this purpose.
  • a test compound may also be modified "in situ" (i.e. once docked into the potential binding domain), enabling immediate evaluation of the effect of replacing selected groups.
  • the computer representation of the test compound may be modified by deleting a chemical group or groups, replacing chemical groups, or by adding a chemical group or groups. After each modification to a compound, the atoms of the modified compound and potential binding site can be shifted in conformation and the distance between the modulator and the active site atoms may be scored on the basis of geometric fit and favourable complementary interactions between the molecules. This technique is described in detail in Molecular Simulations User Manual, 1995 in LUDI. Examples of ligand building and/or searching computer programs include programs in the Molecular
  • the "starting point" for rational ligand design may be a known ligand for the enzyme.
  • a logical approach would be to start with a known ligand (for example a substrate molecule or inhibitor ) to produce a molecule which mimics the binding of the ligand.
  • a known ligand for example a substrate molecule or inhibitor
  • Such a molecule may, for example, act as a competitive inhibitor for the true ligand, or may bind so strongly that the interaction (and inhibition) is effectively irreversible.
  • Such a method may comprise the following steps:
  • the replacement groups could be selected and replaced using a compound construction program which replaces computer representations of chemical groups with groups from a computer database, where the representations of the compounds are defined by structural coordinates.
  • a screening method is provided for identifying a ligand of a galactosyltransferase comprising the step of using the stractural coordinates or model of a substrate molecule or component thereof, defined in relation to its spatial association with a galactosyltransferase stracture or a ligand binding domain, to generate a compound that is capable of associating with the galactosyltransferase or ligand binding domain.
  • the invention contemplates a method for the design of modulators for galactosyltransferases based on the three dimensional stracture or model of a sugar nucleotide donor (or parts thereof) defined in relation to the three dimensional structure of a ligand binding domain.
  • a method for designing potential inhibitors of a galactosyltransferase comprising the step of using the structural coordinates of uracil, uridine, or UDP of Table 5, 6, or 7 to generate a compound for associating with the active site of a ligand binding domain of a galactosyltransferase.
  • a method for designing potential inhibitors of a glycosyltransferase comprising the step of using the structural coordinates of UDP-Gal of Table 6, to generate a compound for associating with the active site of a galactosyltransferase.
  • the following steps are employed in a particular method of the invention: (a) generating a computer representation of UDP-Gal defined by the structural coordinates of Table 6; (b) searching for molecules in a data base that are similar to the defined UDP-Gal using a searching computer program, or replacing portions of the compound with similar chemical stractures from a database using a compound building computer program.
  • the screening methods of the present invention may be used to identify compounds or entities that associate with a molecule that associates with a galactosyltransferase enzyme (for example, a substrate molecule).
  • a galactosyltransferase enzyme for example, a substrate molecule
  • organic compounds may be prepared by organic synthetic methods described in references such as
  • Test compounds and ligands which are identified using a model of the present invention can be screened in assays such as those well known in the art. Screening can be, for example, in vitro, in cell culture, and/or in vivo. Biological screening assays preferably centre on activity-based response models, binding assays (which measure how well a compound binds), and bacterial, yeast and animal cell lines (which measure the biological effect of a compound in a cell). The assays can be automated for high capacity-high throughput screening (HTS) in which large numbers of compounds can be tested to identify compounds with the desired activity. The biological assay, may also be an assay for the ligand binding activity of a compound that selectively binds to the ligand binding domain compared to other enzymes.
  • HTS high capacity-high throughput screening
  • the present invention provides a ligand or compound or entity identified by a screening method of the present invention.
  • a ligand or compound may have been designed rationally by using a model according to the present invention.
  • a ligand or compound identified using the screening methods of the invention specifically associate with a target compound.
  • the target compound may be a galactosyltransferase or a molecule that is capable of associating with a galactosyltransferase (for example a substrate molecule).
  • the ligand is capable of binding to the ligand binding domain of a galactosyltransferase.
  • a ligand or compound identified using a screening method of the invention may act as a "modulator", i.e.
  • a modulator may reduce, enhance or alter the biological function of a galactosyltransferase.
  • a modulator may modulate the capacity of the enzyme to transfer a sugar from a nucleotide sugar donor to a specific hydroxyl of various saccharide acceptors that leads to the formation of a new glycosidic linkage.
  • An alteration in biological function may be characterised by a change in specificity.
  • a modulator may cause the enzyme to accept a different substrate molecule, to transfer a different sugar, or to work with a different metal cofactor. In order to exert its function, the modulator commonly binds to the ligand binding domain.
  • a modulator which is capable of reducing the biological function of the enzyme may also be known as an inhibitor.
  • an inhibitor reduces or blocks the capacity of the enzyme to form new glycosidic linkages.
  • the inhibitor may mimic the binding of a substrate molecule, for example, it may be a substrate analogue.
  • a substrate analogue may be designed by considering the interactions between the substrate molecule and the enzyme (for example by using information derivable from a model of the invention) and specifically altering one or more groups.
  • a modulator acts as an inhibitor of a galactosyltransferase and is capable of inhibiting N- or O-glycan biosynthesis.
  • the present invention also provides a method for modulating the activity of a galactosyltransferase within a cell using a modulator according to the present invention. It would be possible to monitor the expression of N- glycans on the cell surface following such treatment by a number of methods known in the art (for example by detecting expression with an N-and O-glycan specific antibody).
  • the modulator modulates the catalytic mechanism of the enzyme.
  • a modulator may be an agonist, partial agonist, partial inverse agonist or antagonist of a galactosyltransferase or a ligand binding domain.
  • agonist includes any ligand, which is capable of binding to a ligand binding domain and which is capable of increasing a proportion of active enzyme, resulting in an increased biological response.
  • partial agonists includes partial agonists and inverse agonists.
  • partial agonist includes an agonist that is unable to evoke the maximal response of a biological system, even at a concentration sufficient to saturate a specific ligand binding domain.
  • partial inverse agonist includes an inverse agonist that evokes a submaximal response to a biological system, even at a concentration sufficient to saturate a specific ligand binding domain. At high concentrations, it will diminish the actions of a full inverse agonist.
  • the invention relates to a galactosyltransferase ligand binding domain antagonist, wherein said ligand binding domain is that defined by the amino acid structural coordinates described herein.
  • the ligand may antagonise the inhibition of galactosyltransferase by an inhibitor.
  • antagonist includes any agent that reduces the action of another agent, such as an agonist.
  • the antagonist may act at the same site as the agonist (competitive antagonism).
  • the antagonistic action may result from a combination of the substance being antagonised (chemical antagonism) or the production of an opposite effect through a different binding site (functional antagonism or physiological antagonism) or as a consequence of competition for the binding site of an intermediate that links the enzyme to the effect observed (indirect antagonism).
  • competitive antagonism refers to the competition between an agonist and an antagonist for a ligand binding domain that occurs when the binding of agonist and antagonist becomes mutually exclusive. This may be because the agonist and antagonist compete for the same binding site or combine with adjacent but overlapping sites.
  • a third possibility is that different sites are involved but that they influence the same macromolecules in such a way that agonist and antagonist molecules cannot be bound at the same time. If the agonist and antagonist form only short lived combinations with the binding site so that equilibrium between agonist, antagonist and binding site is reached during the presence of the agonist, the antagonism will be surmountable over a wide range of concentrations. In contrast, some antagonists, when in close enough proximity to their binding site, may form a stable covalent bond with it and the antagonism becomes insurmountable when no spare receptors remain.
  • an identified ligand or compound may act as a ligand model (for example, a template) for the development of other compounds.
  • a modulator may be a mimetic of a ligand or ligand binding domain.
  • a mimetic of a ligand may compete with a natural ligand for a galactosyltransferase and antagonize a physiological effect of the enzyme in an animal.
  • a mimetic of a ligand may be an organically synthesized compound.
  • a mimetic of a ligand binding domain may be either a peptide, polysaccharide, oligosaccharide, or other biopharmaceutical (such as an organically synthesized compound) that specifically binds to a natural substrate molecule for a galactosyltransferase and antagonize a physiological effect of the enzyme in an animal.
  • biopharmaceutical such as an organically synthesized compound
  • substitutions may then be made in some of its atoms or side groups in order to improve or modify its binding properties.
  • initial substitutions are conservative, i.e., the replacement group will have approximately the same size, shape, hydrophobicity and charge as the original group. It should, of course, be understood that components known in the art to alter confo ⁇ nation should be avoided.
  • Such substituted chemical compounds may then be analyzed for efficiency of fit to a galactosyltransfease ligand binding domain by the same computer methods described above.
  • positions for substitution are selected based on the predicted binding orientation of a ligand to a galactosyltransferase ligand binding domain.
  • a technique suitable for preparing a modulator will depend on its chemical nature.
  • organic compounds may be prepared by organic synthetic methods described in references such as March, 1994, Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, New York, McGraw Hill.
  • Peptides can be synthesized by solid phase techniques (Roberge JY et al (1995) Science 269: 202-204) and automated synthesis may be achieved, for example, using the ABI 43 1 A Peptide Synthesizer (Perkin Elmer) in accordance with the instructions provided by the manufacturer.
  • the peptide may be purified by preparative high performance liquid chromatography (e.g., Creighton (1983) Proteins Structures and Molecular Principles, WH Freeman and Co, New York NY).
  • the composition of the synthetic peptides may be confirmed by amino acid analysis or sequencing (e.g., the Edman degradation procedure; Creighton, supra).
  • a modulator is a nucleotide, or a polypeptide expressable therefrom, it may be synthesized, in whole or in part, using chemical methods well known in the art (see Caruthers MH et al (1980) Nuc Acids Res Symp Ser 215- 23, Horn T et al (1980) Nuc Acids Res Symp Ser 225-232), or it may be prepared using recombinant techniques well known in the art.
  • Direct synthesis of a ligand or mimetics thereof can be performed using various solid-phase techniques (Roberge JY et al (1995) Science 269: 202-204) and automated synthesis may be achieved, for example, using the ABI 43 1 A Peptide Synthesizer (Perkin Elmer) in accordance with the instructions provided by the manufacturer. Additionally, the amino acid sequences obtainable from the ligand, or any part thereof, may be altered during direct synthesis and/or combined using chemical methods with a sequence from other subunits, or any part thereof, to produce a variant ligand.
  • the coding sequence of a ligand or mimetics thereof may be synthesized, in whole or in part, using chemical methods well known in the art (see Caruthers MH et al (1980) Nuc Acids Res Symp Ser 215-23, Horn T et al (1980) Nuc Acids Res Symp Ser 225-232).
  • host cells can be employed for expression of the nucleotide sequences encoding a ligand of the present invention. These cells may be both prokaryotic and eukaryotic host cells. Suitable host cells include bacteria such as E. coli, yeast, filamentous fungi, insect cells, mammalian cells, typically immortalized, e.g., mouse, CHO, human and monkey cell lines and derivatives thereof. Preferred host cells are able to process the expression products to produce an appropriate mature polypeptide. Processing includes but is not limited to glycosylation, ubiquitination, disulfide bond formation and general post-translational modification.
  • the ligand may be a derivative of, or a chemically modified ligand.
  • derivative or “derivatised” as used herein includes the chemical modification of a ligand.
  • a chemical modification of a ligand and/or a key amino acid residue of a ligand binding domain of the present invention may either enhance or reduce hydrogen bonding interaction, charge interaction, hydrophobic interaction, Van Der Waals interaction or dipole interaction between the ligand and the key amino acid residue(s) of a galactosyltransferase ligand binding domain.
  • modifications involve the addition of substituents onto a test compound such that the substituents are positioned to collide or to bind preferentially with one or more amino acid residues that correspond to the key amino acid residues of a galactosyltransferase ligand binding domain.
  • Typical modifications may include, for example, the replacement of a hydrogen by a halo group, an alkyl group, an acyl group or an amino group.
  • the invention also relates to classes of modulators of galactosyltransferase based on the stracture and shape of a substrate, defined in relation to the substrate's molecule's spatial association with a galactosyltransferase structure of the invention or part thereof. Therefore, a modulator may comprise a substrate molecule having the shape or structure, preferably the stractural coordinates, of a substrate molecule in an active site binding pocket of a reaction catalyzed by a galactosyltransferase. Modulators Based on the 3D Structure of a Nucleotide Sugar Donor
  • One class of modulators defined by the invention are compounds of the following formula I having the structural coordinates of uracil of Table 5, preferably Run 9, Cluster 1 or ATOM 1 to ATOM 9, inclusive of Table 7:
  • Ri and R 2 are each independently hydrogen, alkyl, cycloalkyl, alkenyl, alkynyl, heterocyclic rings, aryl, alkoxy, aryloxy, hydroxyl, thiol, thioaryl, amino, halogen, carboxylic acid or esters or thioesters thereof, amines, sulfate, sulfonic or sulf ⁇ nic acid or esters thereof, phosphate, pyrophate, gallic acid, phosphonates, thioamide, and -OR ⁇ 2 where R 12 is alkyl, cycloalkyl, alkenyl, alkynyl, or heterocyclic ring; and salts and optically active and racemic forms of a compound of the formula I.
  • modulators defined by the invention are compounds of the following formula II having the stractural coordinates of uridine of Table 5, preferably Run 9, Cluster 1 or ATOMs 1 to 20 inclusive, of Table 7:
  • R ls R 2 , R 3 , R 4 , and R 5 are each independently hydrogen, alkyl, cycloalkyl, alkenyl, alkynyl, heterocyclic rings, aryl, alkoxy, aryloxy, hydroxyl, thiol, thioaryl, amino, halogen, carboxylic acid or esters or thioesters thereof, amines, sulfate, sulfonic or sulfinic acid or esters thereof, phosphate, pyrophosphate, gallic acid, phosphonates, thioamide, and -OR 12 where Rj 2 is alkyl, cycloalkyl, alkenyl, alkynyl, or heterocyclic ring, and salts and optically active and racemic forms of a compound of the formula II.
  • Yet another class of modulators defined by the invention are compounds of the following formula III having the stractural coordinates of UDP in Table 5, preferably Run 9, Cluster 1, or ATOMs 1 to 28 inclusive of
  • R-, R 2 , R 3 , R-, R ⁇ , and R ⁇ are each independently hydrogen, alkyl, cycloalkyl, alkenyl, alkynyl, heterocyclic rings, aryl, alkoxy, aryloxy, hydroxyl, thiol, thioaryl, amino, halogen, carboxylic acid or esters or thioesters thereof, amines, sulfate, sulfonic or sulfinic acid or esters thereof, phosphate, gallic acid, phosphonates, thioamide, and -OR 12 where R !2 is alkyl, cycloalkyl, alkenyl, alkynyl, or heterocyclic ring, Re may be a monosaccharide or disaccharide, preferably a monosaccharide, including galactose, glucose, and mannose, and salts and optically active and racemic forms of a compound of the formula III.
  • Yet another class of modulators defined by the invention are compounds of the following formula IV having the structural coordinates of UDP-Gal in Table 6, preferably Run, Cluster 1:
  • Ri, R 2 , R 3 , R 4 , R 7 , R 8 , R 9 , and R I0 are each independently hydrogen, alkyl, cycloalkyl, alkenyl, alkynyl, heterocyclic rings, aryl, alkoxy, aryloxy, hydroxyl, thiol, thioaryl, amino, halogen, carboxylic acid or esters or thioesters thereof (e.g.
  • R ]2 is alkyl, cycloalkyl, alkenyl, alkynyl, or heterocyclic ring
  • X is a counter-ion including sodium, lithium, potassium, calcium, magnesium, manganese, cobalt ions and the like, as well as nontoxic ammonium, quaternary ammonium, and amine cations, preferably Mn 2+ , and salts and optically active and racemic forms of a compound of the formula IV.
  • R b R 2 , R3, R4, R5, Re, R 7 , Rs, R 9 , and/or R* 0 alone or together, which contain available functional groups as described herein, may be substituted with for example one or more of the following: alkyl, alkoxy, hydroxyl, aryl, cycloalkyl, alkenyl, alkynyl, thiol, thioalkyl, thioaryl, amino, or halo.
  • the term "one or more" used herein preferably refers to from 1 to 2 substituents.
  • the present invention contemplates all optical isomers and racemic forms thereof of the compounds of the invention, and the formulas of the compounds shown herein are intended to encompass all possible optical isomers of the compounds so depicted.
  • the present invention also contemplates salts and esters of the compounds of the invention.
  • the present invention includes pharmaceutically acceptable salts.
  • pharmaceutically acceptable salts are meant those salts which are suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response and the like, and are commensurate with a reasonable benefit/risk ratio.
  • Pharmaceutically acceptable salts are well known in the art and are described for example, in S. M. Berge, et al., J. Pharmaceutical Sciences, 1977, 66:1-19. Compositions and Methods of Treatment
  • the ligands and the modulators of the invention e.g.
  • inhibitors may be used to modulate the biological activity of a galactosyltransferase in a cell, including modulating a pathway in a cell regulated by the galactosyltransferase or modulating a galactosyltransferase with inappropriate activity in a cellular organism.
  • the present invention thus provides a method for treating a condition in a subject regulated by a galactosyltransferase or involving inapproproriate galactosyltransferase activity comprising administering to a subject an effective amount of a modulator identified using the methods of the invention.
  • the invention still further relates to a pharmaceutical composition which comprises a three dimensional galactosyltransferase of the invention or a portion thereof (e.g. a ligand binding domain), or a modulator of the invention in an amount effective to regulate one or more of the above-mentioned conditions and a pharmaceutically acceptable carrier, diluent or excipient.
  • the invention also provides the use of a ligand or modulator according to the invention in the manufacture of a medicament to treat and/or to prevent a disease in a patient.
  • Inhibitors or antagonists of ⁇ l,3-Gal fransferase of the present invention may be particularly useful in reducing xenotransplant rejection in an animal patient.
  • Xenograft tissue may be treated with, or derived from an animal that has been treated with an inhibitor to decrease Gal ⁇ (l,3) Gal epitopes on the xenograft tissue. This treatment will reduce or avoid an immune reaction between circulating antibodies in the transplant recipient reactive with the epitopes.
  • the xenograft tissue is of pig origin and the xenograft recipient is a human.
  • the xenograft tissue includes any tissue which expresses antigens having GaI ⁇ (l,3)Gal epitopes.
  • the tissue may be in the form of an organ, for example a kidney, heart, lung, or liver, or it may be in the form of parts of organs, cell clusters, glands and the like (e.g. lenses, pancreatic islet cells, skin, and corneal tissue).
  • organs for example a kidney, heart, lung, or liver
  • parts of organs e.g. lenses, pancreatic islet cells, skin, and corneal tissue.
  • the modulators of the invention may be converted using customary methods into pharmaceutical compositions.
  • the pharmaceutical compositions contain the modulators either alone or together with other active substances.
  • Such pharmaceutical compositions can be for oral, topical, rectal, parenteral, local, inhalant, or intracerebral use. They are therefore in solid or semisolid form, for example pills, tablets, creams, gelatin capsules, capsules, suppositories, soft gelatin capsules, liposomes (see for example, U.S. Patent Serial No. 5,376,452), gels, membranes, and tubelets.
  • those forms for intramuscular or subcutaneous administration can be used, or forms for infusion or intravenous or intracerebral injection can be used, and can therefore be prepared as solutions of the modulators or as powders of the modulators to be mixed with one or more pharmaceutically acceptable excipients or diluents, suitable for the aforesaid uses and with an osmolarity which is compatible with the physiological fluids.
  • those preparations in the form of creams or ointments for topical use or in the form of sprays should be considered; for inhalant uses, preparations in the form of sprays should be considered.
  • the pharmaceutical compositions can be prepared by per se known methods for the preparation of pharmaceutically acceptable compositions which can be administered to patients, and such that an effective quantity of the active substance is combined in a mixture with a pharmaceutically acceptable vehicle.
  • Suitable vehicles are described, for example, in Remington's Pharmaceutical Sciences (Remington's Phannaceutical Sciences, Mack Publishing Company, Easton, Pa., USA 1985).
  • the pharmaceutical compositions include, albeit not exclusively, the modulators in association with one or more pharmaceutically acceptable vehicles or diluents, and contained in buffered solutions with a suitable pH and iso-osmotic with the physiological fluids.
  • the modulators may be indicated as therapeutic agents either alone or in conjunction with other therapeutic agents or other forms of treatment.
  • inhibitors may be used in combination with anti-proliferative agents, antimicrobial agents, immunostimulatory agents, or anti-inflammatories.
  • the modulators may be administered concurrently, separately, or sequentially with other therapeutic agents or therapies.
  • compositions containing modulators can be administered for prophylactic and/or therapeutic treatments.
  • compositions are administered to a patient already suffering from a condition as described above, in an amount sufficient to cure or at least alleviate the symptoms of the disease and its complications.
  • An amount adequate to accomplish this is defined as a "therapeutically effective dose”. Amounts effective for this use will depend on the severity of the disease, the weight and general state of the patient, the nature of the administration route, the nature of the formulation, and the time or interval at which it is administered.
  • compositions containing modulators are administered to a patient susceptible to or otherwise at risk of a particular condition.
  • Such an amount is defined to be a "prophylactically effective dose”.
  • the precise amounts depend on the patient's state of health and weight, the nature of the administration route, the nature of the formulation, and the time or interval at which it is administered.
  • Example 1 The following non-limiting examples illustrate the invention: Example 1
  • the modeling of bovine ⁇ -l,3-GalT was carried out using homology modeling procedures and ⁇ -l,3-GalT- ligand complexes were generated using automated docking procedures. These computational modeling approaches allow fairly reasonable predictions of three-dimensional stractures of proteins and their complexes with substrates and ligands thereby offering a rational way of investigating structure-function relationships (12).
  • the amino acid sequence of ⁇ -l,3-GalT was obtained from a publicly available sequence data bank (13). Homology modeling. - The basic steps in the construction of a protein model based on a homologous structure are sequentially in the following order: amino acid sequence alignment, copying aligned coordinates, building loops, and refinement.
  • sequence alignment and secondary stracture predictions were carried out using the Fold recognition server located at UCLA (14).
  • the Molecular Simulations Inc. collection of programs was used for all protein modeling (15-17).
  • the template stracture chosen was the three-dimensional crystal structure (9) of SpsA determined at a resolution of 1.5 A.
  • the initial alignment of ⁇ -l,3-GalT and SpsA fransferase sequences was obtained using the pair-wise alignment with the HOMOLOGY program (15).
  • Multiple alignment of amino acid sequences was performed using the Needleman and Wunch method (18). This method is capable to provide an optimum alignment of two sequences that represents the best overall balance between the number of good amino acid matches and the least number of required gaps.
  • the initial pair-wise sequence alignments were manually modified to obtain structure-oriented alignments.
  • die coordinates of the homologous regions were transferred from the SpsA structure to the bovine ⁇ -l,3-GalT using the MODELER program (16).
  • the geometry of the generated model was then locally optimized to remove steric side-chain clashes.
  • the builder module of the Insightll program (17) was used to add hydrogen atoms to the enzyme and assign partial charges. Docking.
  • a Mn 2+ cation position was located, based on the SpsA structure, near the side chain of the Asp227, which belongs to the aspartate-valine-aspartate (DVD) sequence motif.
  • An aspartate-any residue-aspartate (DXD) or the aspartate-any residue-histidine (DXH) motif is common to many glycosyl transferases (21) and is involved in binding metal cations as well as its substrate. Water molecules were not considered in these computations. Positions of all protein atoms were fixed during the docking. The dihedral angles of all ligands were optimized while bond lengths and bond angles were restrained to standard values.
  • Table 3 shows the predicted secondary structures for the ⁇ - 1,3-GalT sequence that was used for generating a homology model of ⁇ -l,3-GalT.
  • the homology model of ⁇ -l,3-GalT consists of two compact domains.
  • the predicted N-terminal domain has about 100 residues starting at Gin- 125 and ends at Gln-231 and the C-terminus domain has the remaining modeled residues.
  • Figure 2 shows a superposition of the ⁇ -l,3-GalT model (blue) and the corresponding SpsA structure (magenta).
  • the amino acid residues of SpsA that interact with the UDP ligand are shown as tubes.
  • the corresponding amino acid residues of ⁇ -l,3-GalT are shown as thin tubes.
  • the active site consists of an open ⁇ , ⁇ -sandwich made up of three helices packed against four standard ⁇ - sheets.
  • the general topology of the modeled ⁇ -l,3-GalT resembles those of GnT I and SpsA with the secondary structural elements similarly arranged in space.
  • the following amino acid residues have been identified to be part of the UDP docking pocket of ⁇ -l,3-GalT: Phe-134, Tyr-139, IIe-140, Val-136, Arg-194, Arg-202, Lys-209, Asp-173, His-218, Thr-137, Asp-225, Val-226, and Asp-227.
  • the modeled catalytic domain has a core structure common to most of the known transferases (9-11).
  • amino acid residues that are involved in the UDP-Gal recognition and in the catalytic mechanism are homologous both in sequence and spatial relationship.
  • the overall electrostatic property of the active site of the ⁇ -l,3-GalT is highly comparable with the UDP binding sites of GnTI and SpsA.
  • the present analysis suggests that although the sequence homologies of SpsA, GnT I and ⁇ - 1,3-GalT are relatively low, they have a structurally conserved framework of about 100 residues that specifically recognize UDP.
  • the ribose ring packs with the conserved hydrophobic residue (Thr-9 in SpsA and Ile-113 in GnT I) that is located at the bottom of the pocket.
  • the metal binding site is located at one of the ⁇ -strands that contains the conserved DVD (Asp-225, Val-226 and Asp227) motif.
  • These conserved residues are assumed to be located in the vicinity of the pyrophosphate-binding region.
  • the C-terminal portion of the model has a confined groove, which has a stretch of charged residues. The docking studies described below suggest that this region can specifically recognize inhibitors, which are designed based on the acceptor substrate model (19).
  • UDP binds in the deep pocket generally in a similar conformation.
  • Three hydrogen bonds that are possible between the uracil and ⁇ -1,3- GalT characterize this binding mode. These are (1) the amide hydrogen of uracil in position 3 and OD1 of Asp-168, (2) the carbonyl oxygen of uracil in position 4 and the side chain of Lys-204, and (3) the carbonyl oxygen of uracil in position 2 and the amide hydrogen of the His-213 side chain.
  • the hydroxyl groups at the 2 and 3 positions of the ribose ring forms three hydrogen bonds with the Asp-225 side chain oxygens.
  • the pyrophosphate oxygens interact with the Asp-227 side chain through the metal ion. Apart from these hydrogen bond interactions many favorable hydrophobic interactions are possible between the uridine and the protein. It is clear from Table 1 that the bound UDP generally favors interactions with conserved amino acid residues of the enzyme. However, some of the residues that do not interact directly with UDP but lie in the close vicinity of the UDP docked region are Tyr-139, IIe-140, Val- 136, Arg-194, Asp-197, Ile-198, Arg-202, Lys-204, His209 and His-213. It is noteworthy that some of these residues such as Tyrl39, Asp-197 are conserved across various species (8). It is possible that these active site side chains may be involved in direct binding interactions with UDP.
  • the lowest energy cluster consists of about 30% of all the docking runs.
  • the analysis of the other low energy clusters that represent about 70% of docked structures clearly shows that many of the docking modes were very close to the lowest energy-binding mode. However, small variations in the nature of local interactions between the pyrophosphate part and the enzyme were observed. It can be seen from Figure 3 that the 5 and 6 positions of the uracil ring are exposed to the solvent and the remaining positions of the uracil fragment are in contact with the protein.
  • the structure of the UDP-Gal complex with ⁇ -l,3-GalT has been generated using the approach described above.
  • Figure 4 shows the low energy binding modes of this complex.
  • the UDP is bound at the active site of the enzyme (8).
  • the uracil ring of the bound UDP is placed into the cavity where its carbonyl and amide hydrogens form two hydrogen bonds with side-chains of Arg-71 and Asp-39, respectively. Apart from these hydrogen bond interactions, a favorable stacking interaction between the uracil ring and side chain of Tyr-11 is possible.
  • a strong hydrogen bond interaction is possible between the hydroxyl of ribose in the position 3 and the side chain oxygen of Asp-99.
  • the pyrophosphate conformation is confined to a particular orientation due to the favorable charge interactions with the bound metal ion.
  • Unligil et al (10) has solved a structure of GnT I complexed with UDP-GlcNAc at 1.5 A resolution.
  • the uracil ring favors a similar interaction, as observed in the SpsA complex, with the nucleotide binding domain residues consisting of a Lys and an Asp.
  • the ribose portions of the UDP bind into the hydrophobic rich region of the GnT I and thereby gains a stacking energy.
  • these two structures possess a clear structural and sequence similarity at the UDP binding pocket. However, overall there is no sequence homology between the two proteins.
  • the bound UDP conformation is very similar in these stractural complexes.
  • Binding mode of an inhibitor to a-l,3-GalT Recently, an inhibitor based on the acceptor of ⁇ - 1,3 -GalT has been designed (19). This compound has a disaccharide linked to a bromine substituted naphthamide ring. It has been shown that the removal of the terminal sugar unit in this inhibitor does not inhibit ⁇ -l,3-GalT, but instead inhibits ⁇ -l,4-GalT. Thus, the determination of the bindmg mode of this inhibitor to ⁇ - 1,3 -GalT might provide a stereochemical explanation for the observed binding affinities. Using the above described docking procedure, this synthetic inhibitor was docked to the surface of ⁇ -1,3- GalT.
  • FIG. 5 shows the computed binding mode of the inhibitor at the acceptor-binding region of the protein.
  • the terminal saccharide binds close to the Asp-227 side chain and the bulky aromatic group of the inhibitor interacts with the side chain of of Ile-283.
  • the bromide atom is located close to the side chain of Asp-227 and the naphthamide ring is placed on the top of Met-224 side chain. It can be seen that the inhibitor not only occupies the acceptor-binding region of the protein but also has considerable interactions at the donor site of the enzyme. Thus, these predicted binding modes of inhibitor could explain its inhibitory activity.
  • Figures 6 to 9 also show models of ⁇ -l,3-GalT and ligand binding domains of the enzyme.
  • the ⁇ -l,3-GalT structure and its complexes with UDP, UDP-Gal, and a synthetic inhibitor have been modeled.
  • the predicted N-terminal domain of the of the ⁇ -l,3-GalT has about 100 residues that start at Gln-125 and end at Gln-131.
  • the overall secondary structure arrangements, amino acid properties, spatial arrangement of critical amino acid residues and size of this domain are highly comparable with other GnT structures.
  • the predicted pocket on this domain surface of ⁇ - 1,3-GalT specifically recognizes UDP in a unique binding mode.
  • Needlman SB Wunsch CD. A general method applicable to the search for similarities in the amino acid sequence of two proteins. J Mol Biol 1970; 48: 443-453.
  • ACCESSIBILITY 3st P_3 acc be ee ebbeeee lOst: PHD acc 0657736006799 Rel acc 1206411242333 subset: SUB acc .. , ee.. ,b..
  • ATOM 194 CA GLY 141 14. ,425 15. ,788 22. .033
  • ATOM 234 HD2 TYR 143 13.633 12.650 28.365
  • ATOM 258 HD1 ILE 144 11.225 21.711 25.940
  • ATOM 281 CA HIS 146 5. ,227 14, .935 24, .880
  • ATOM 282 HA HIS 146 4, .330 14, .892 24, .262
  • ATOM 284 HD1 HIS 146 4. ,262 12, .665 27, .699
  • ATOM 294 HB2 HIS 146 6. .040 12, .924 24, .443
  • ATOM 300 HA TYR 147 5. .026 16, .397 28, .707
  • ATOM 302 HB1 TYR 147 7, .620 17, .877 28, .183
  • ATOM 306 HD1 TYR 147 5. .019 17. .237 30. .798
  • ATOM 308 HD2 TYR 147 7. ,220 20. .183 28. .649
  • ATOM 329 HD2 LEU 148 3. ,712 21. ,169 22. ,945
  • ATOM 332 HD1 LEU 148 6, ,002 23. ,199 22. ,670
  • ATOM 334 HD1 LEU 148 6. ,242 21. ,452 22. ,427
  • N3 N3 tS3 [ ⁇ ) W t ⁇ 3 N3 N- M !N3 [ ⁇ N3 N3 [ ⁇ W N3 W K3 [NJ H H H* f- J h J H-* P J h J l J h J I ⁇ H H
  • ATOM 435 CA ALA 155 -8, .627 20, .913 29 .284
  • ATOM 436 HA ALA 155 -8, .526 21, .664 28, .501
  • ATOM 438 HB1 ALA 155 ⁇ 10, .693 20, .467 28 .833
  • ATOM 442 O ALA 155 -9. .412 22. .669 30. .649
  • ATOM 445 CA ASN 156 -8, .972 21. .225 33. .022
  • ATOM 453 HD2 ASN 156 -5. ,527 23. ,497 32. ,199
  • ATOM 454 HD2 ASN 156 -7. ,269 23. ,874 32. ,120
  • ATOM 456 O ASN 156 • 11. ,114 22. ,056 32. ,428
  • ATOM 618 CD1 ILE 166 4, .406 24 .346 30, .555
  • ATOM 619 HD1 ILE 166 5, .263 23, .705 30, .765
  • ATOM 620 HD1 ILE 166 3, .569 24, .050 31, .187
  • ATOM 621 HD1 ILE 166 4, .124 24, .244 29, ,507
  • ATOM 626 CA PHE 167 7. .217 29, .573 30, .535
  • ATOM 633 HD1 PHE 167 6, .250 31, .539 32. .919
  • ATOM 636 CE1 PHE 167 7. .569 31, .660 34. .590
  • ATOM 646 CA TYR 168 8. ,154 26, .252 29. .272
  • ATOM 652 CD1 TYR 168 6. .214 24. .010 28, .059
  • ATOM 653 HD1 TYR 168 6. .734 23. ,563 28. .907
  • ATOM 655 HD2 TYR 168 6. .631 26, .412 25. ,744
  • ATOM 670 HB ILE 169 11. .252 25. ,950 26. 085
  • ATOM 679 HD1 ILE 169 14. ,673 24. ,944 25. 231 ⁇ , C ⁇ C ⁇ to to
  • ATOM 770 CA ARG 176 19, .777 21 .686 17 .628
  • ATOM 780 HD2 ARG 176 22, .867 20, .438 15. .345
  • ATOM 810 CA PRO 178 15. ,853 19. .648 20. .824
  • ATOM 812 CD PRO 178 17. ,943 18. .564 20. ,396
  • ATOM 814 HD2 PRO 178 17. ,955 17. ,611 19. ,868
  • ATOM 825 CA LEU 179 15. .528 23 .241 19, .724
  • ATOM 826 HA LEU 179 15. .187 23, .666 20, .668
  • ATOM 830 CG LEU 179 17, ,890 24, .305 19, .845
  • ATOM 833 HD2 LEU 179 18. .579 2 .896 21, .804
  • ATOM 834 HD2 LEU 179 17, .116 25 .748 21 .253
  • ATOM 835 HD2 LEU 179 17, .009 24, .058 21. .801
  • ATOM 837 HD1 LEU 179 19, .794 25 .312 19 .674
  • ATOM 838 HDl LEU 179 19, .119 24, .752 18, .125
  • ATOM 844 CA ILE 180 13. .609 22, .175 16. ,662
  • ATOM 845 HA ILE 180 13, .363 23, .164 16. .275
  • ATOM 847 HB ILE 180 13, .225 21 .314 14, .767
  • ATOM 856 HDl ILE 180 14. .570 17. .918 15. ,043
  • ATOM 857 HDl ILE 180 13. .517 18. .977 14. ,076
  • ATOM 858 HDl ILE 180 15. .276 19. ,245 14. .090
  • ATOM 860 O ILE 180 11. ,264 21. .896 16. ,861
  • ATOM 863 CA GLU 181 11. .440 19, .864 18. ,692
  • ATOM 872 ' OE1 GLU 181 11. .057 16. ,057 19. ,201
  • ATOM 876 O GLU 181 9. .297 20. .797 19. ,038
  • ATOM 879 CA LEU 182 10. .104 22, .736 20. ,697
  • ATOM 880 HA LEU 182 9. .331 22, .163 21. ,209
  • ATOM 882 HB1 LEU 182 10. ,048 24. .499 22. ,015
  • ATOM 883 HB2 LEU 182 11. ,640 24. .149 21. .399

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Abstract

L'invention concerne des structures et des modèles de domaines de liaison aux ligands de galactosyltransférases, et les domaines de liaison avec les ligands. Les coordonnées structurelles qui définissent les structures et n'importe quels ligands fixés aux structures permettent la détermination d'homologues, des structures de polypeptide qui étaient inconnues, et l'identification de modulateurs des galactosyltransférases. L'invention concerne également des structures et des modèles de donneurs de sucre nucléotidiques pour les galactosyltransférases, ainsi que la conception de modulateurs pour les galactosyltransférases, fondée sur les propriétés de ces structures et modèles.
PCT/CA2001/000607 2000-05-03 2001-05-02 Conception de modulateurs pour galactosyltransferases Ceased WO2001083717A2 (fr)

Priority Applications (1)

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AU2001256030A AU2001256030A1 (en) 2000-05-03 2001-05-02 Designing modulators for alpha-1, 3 galactosyltransferases based on a structural model

Applications Claiming Priority (2)

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US20184100P 2000-05-03 2000-05-03
US60/201,841 2000-05-03

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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002059715A3 (fr) * 2000-12-29 2003-07-31 Triad Therapeutics Inc Procedes pour predire des proprietes fonctionnelles et structurales de polypeptides au moyen de modeles de sequences
US7811781B2 (en) 2005-07-06 2010-10-12 Btg International Limited Core 2 β(1,6)-acetylglycosaminyltransferase as diagnostic marker for atherosclerosis
CN101875697A (zh) * 2010-04-15 2010-11-03 北京天广实生物技术股份有限公司 新型抗egfr人源抗体tgm10的设计及其应用
US7906493B2 (en) 2003-12-22 2011-03-15 Btg International Limited Core 2 GlcNAc-T inhibitors
US7998943B2 (en) 2005-07-06 2011-08-16 Btg International Limited Core 2 GlcNAc-T inhibitors III
US8197794B2 (en) 2003-12-22 2012-06-12 Ms Therapeutics Limited Core 2 GlcNAc-T inhibitors
US8609633B2 (en) 2005-07-06 2013-12-17 Ms Therapeutics Limited Core 2 GlcNAc-T inhibitors
CN116144682A (zh) * 2023-03-23 2023-05-23 石河子大学 一种羽毛针禾棉子糖合酶基因SpRAFS1在促进植物根发育中的应用

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5849991A (en) * 1994-01-27 1998-12-15 Bresatch Limited Mice homozygous for an inactivated α 1,3-galactosyl transferase gene

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002059715A3 (fr) * 2000-12-29 2003-07-31 Triad Therapeutics Inc Procedes pour predire des proprietes fonctionnelles et structurales de polypeptides au moyen de modeles de sequences
US7906493B2 (en) 2003-12-22 2011-03-15 Btg International Limited Core 2 GlcNAc-T inhibitors
US8197794B2 (en) 2003-12-22 2012-06-12 Ms Therapeutics Limited Core 2 GlcNAc-T inhibitors
US7811781B2 (en) 2005-07-06 2010-10-12 Btg International Limited Core 2 β(1,6)-acetylglycosaminyltransferase as diagnostic marker for atherosclerosis
US7998943B2 (en) 2005-07-06 2011-08-16 Btg International Limited Core 2 GlcNAc-T inhibitors III
US8609633B2 (en) 2005-07-06 2013-12-17 Ms Therapeutics Limited Core 2 GlcNAc-T inhibitors
CN101875697A (zh) * 2010-04-15 2010-11-03 北京天广实生物技术股份有限公司 新型抗egfr人源抗体tgm10的设计及其应用
CN101875697B (zh) * 2010-04-15 2014-04-30 北京天广实生物技术股份有限公司 新型抗egfr人源抗体tgm10的设计及其应用
CN116144682A (zh) * 2023-03-23 2023-05-23 石河子大学 一种羽毛针禾棉子糖合酶基因SpRAFS1在促进植物根发育中的应用
CN116144682B (zh) * 2023-03-23 2024-04-12 石河子大学 一种羽毛针禾棉子糖合酶基因SpRAFS1在促进植物根发育中的应用

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AU2001256030A1 (en) 2001-11-12

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