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WO2007039761A2 - Structure cristalline de cd44 et son utilisation - Google Patents

Structure cristalline de cd44 et son utilisation Download PDF

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Publication number
WO2007039761A2
WO2007039761A2 PCT/GB2006/003734 GB2006003734W WO2007039761A2 WO 2007039761 A2 WO2007039761 A2 WO 2007039761A2 GB 2006003734 W GB2006003734 W GB 2006003734W WO 2007039761 A2 WO2007039761 A2 WO 2007039761A2
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atom
ligand
tyr
hoh
thr
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PCT/GB2006/003734
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WO2007039761A3 (fr
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David G. Jackson
Suneale Banerji
Martin Noble
Anthony J. Day
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Medical Research Council
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6872—Intracellular protein regulatory factors and their receptors, e.g. including ion channels
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70585—CD44
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2299/00—Coordinates from 3D structures of peptides, e.g. proteins or enzymes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/70585—CD44
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value

Definitions

  • the present invention relates to the crystal structure of a co-complex of the protein CD44 and hyaluronan (HA), and uses thereof.
  • the hyaluronan receptor CD44 mediates cell adhesion and migration through pericellular matrix during the processes of embryonic morphogenesis and leukocyte homing. It has become increasingly clear that such interactions are particularly important to the pathology of inflammation and cancer and consequently CD44 is an attractive target for therapeutic drug development.
  • CD44-ligand interaction is currently unknown. It has previously been shown that the HA-binding domain comprises an extended form of the C-type lectin like Link module in which C- and N-terminal extensions form an additional structural lobe that is obligatory for ligand-binding.
  • the making and breaking of multiple weak binding interactions between cells and components of the extracellular matrix permits migration within the tissues during such diverse processes as embryonic morphogenesis, wound healing and inflammatory tissue homing (1 , 2)
  • One ubiquitous component of the matrix that is central to these processes is the glycosaminoglycan hyaluronan, a high molecular weight co-polymer of N-acetylglusoamine and glucuronic acid (GlcNAc ⁇ i -4GIcUA) n whose hygroscopic properties maintain low resistance pathways for cell movement in mesenchymal and other tissues.
  • hyaluronan is not covalently attached to a polypeptide core but exists as an independent non-branched polysaccharide that associates non-covalently with large matrix proteoglycans of the Link protein superfamily such as aggrecan, versican and link protein in the extracellular matrix.
  • the interaction of cells with these protein-bound HA complexes and their subsequent migration is mediated primarily by the CD44 molecule, the major transmembrane receptor of the Link protein superfamily.
  • the CD44 molecule functions as a tissue homing receptor on inflammatory leukocytes. Functionally silent on resting leukocytes, CD44 becomes competent to bind HA after prior activation by pro-inflammatory cytokines (3-5). Thus, discrete populations of cells in peripheral blood are directed to extravasate at inflammatory sites, where they are thought to "roll" on HA displayed on the luminal surface of activated endothelial cells prior to transmigration (see eg. (3)).
  • CD44 Crohn's disease
  • CD44 was shown to be functionally associated in leukocytes with another homing receptor, the ⁇ 4 ⁇ 1 integrin VLA-4 - a ligand for the adhesion molecule VCAM-1 that mediates firm adhesion of lymphocytes and monocytes in inflamed vessel endothelium (14).
  • CD44-mediated rolling on HA may therefore precede stable adhesion to endothelium via VLA-4/VCAM-1 in much the same way as selectin- mediated rolling on mucins.
  • ⁇ 4 integrin is currently a major target for antibody-based drug therapy.
  • CD44 represents an attractive target for antibody therapy.
  • CD44 would seem particularly suited to structure-based anti-inflammatory drug design.
  • Link module a conserved unit of approximately 100 residues, comprising two antiparallel ⁇ -sheets made from a total of six individual ⁇ strands ( ⁇ 1- ⁇ 6) and two short ⁇ -helices that form a beta- fold, stabilized by two highly conserved disulfide bridges.
  • Link module is structurally related to the carbohydrate-binding fold in C-type lectins, although the two gene families share no significant amino acid sequence homology (16).
  • the single Link module represents an elaboration of the basic consensus unit to form an enlarged and extended structure.
  • the CD44 HA-binding domain has the six ⁇ strands of the consensus Link module ( ⁇ 1- ⁇ 6) extended by four additional ⁇ strands (denoted 0, 7, 8 and 9), coming from the N- and C-terminal extensions, which form a distinct structural lobe (19).
  • the Link extension carries two of the sites for covalent attachment of N-glycan chains whose terminal sialylation blocks HA-binding, perhaps as a result of steric hindrance.
  • the present invention provides the co-crystallization of the murine CD44 with its ligand HA and elucidation of the 3D structure of the CD44:HA complex. This has revealed for the first time the molecular basis for HA-binding to a cell surface receptor and provides the necessary information for the development of small molecule inhibitors for the treatment of inflammatory and neoplastic diseases.
  • Another surprising feature of the present invention is the finding that the residue Arg45
  • binding groove for HA is surprisingly shallow and that the interactions between CD44 and HA are predominantly hydrogen bonds rather than ionic.
  • the unusual nature of the binding groove in CD44 may therefore have evolved to permit weak engagement with ligand, consistent with the role of the receptor in making reversible low affinity interactions with large HA polymers during cell migration.
  • the present invention relates to the crystal structure of a co-complex of CD44 and HA, which has identified novel interactions between HA and the CD44 receptor.
  • the present inventors have obtained a novel co-crystal of CD44 with HA which is useful for the provision of atomic co-ordinate models of this protein.
  • the models may be used in modelling the interaction of ligands with CD44, e.g. in the development of novel CD44-binding molecules or in the development or modification of existing CD44-binding molecules.
  • the invention provides a three dimensional structure of CD44 set out in Tables 1 2, 4 or 5, and uses of these as well as the CD44 structure of Table 3.
  • novel findings may be used to model the interaction of HA, and other ligands or potential ligands, to other protein structures which contain a Link module domain.
  • Table 1 provides the co-ordinate data of the form A co-crystal of mouse CD44, in complex with HA.
  • Table 2 provides the co-ordinate data of the form B co-crystal of mouse CD44, in complex with HA.
  • Table 3 provides an apo structure of human CD44.
  • Table 4 provides a model of form B co-crystal of human CD44, in complex with HA.
  • Table 5 provides a homology model of Lyve-1 , derived from the structure of Table 1.
  • Table 6 sets out the atoms of the contact residues involved in HA-binding.
  • FIG. 1 Alignment of Mouse CD44 1-183 (SEQ ID NO:1) vs. Human CD44 1-178 (SEQ ID NO:2) vs. LYVE-1 29-142 (SEQ ID NO:3). Residues of these molecules in the present specification are referred to by the numbering shown in this Figure.
  • Such a co-crystal may be obtained using the methods described in the accompanying examples.
  • the invention thus further provides a CD44-HA co-crystal having a resolution of at least 1.3 A.
  • the invention also provides a co-crystal of CD44-HA having the three- dimensional atomic co-ordinates of Table 1 or 2.
  • An advantageous feature of the structures defined by the atomic co-ordinates of Table 1 and 2 is that they have a resolution of about 1.3A, allowing resolution of the contacts between HA and residues of the CD44 to be resolved and identified.
  • Tables 1 and 2 give atomic co-ordinate data for the A and B forms of the co-crystal of CD44- HA.
  • the third column denotes the atom, the fourth the residue type, the fifth the chain identification (in this case, chain A for CD44 and C for the HA), the sixth the residue number, the seventh, eighth and ninth columns are the X, Y, Z co-ordinates respectively of the atom in question, the tenth column the occupancy of the atom, the eleventh the temperature factor of the atom, the twelfth the atom type.
  • Ligand and solvent molecules are also included in the Table, the former as "C" chain atoms.
  • Table 3 provides atomic co-ordinate data for human CD44. This is pdb file 1 UUH. This is in a similar format to Table 1 , though comprises two copies of the CD44 protein, designated A chain and B chain. Solvent molecules are given chain identifiers to indicate the protein chain to which they are most closely associated with, where A chain solvent molecules are designated Z and B chain solvent molecules are designated Y. Reference to Table 3, selected co-ordinates thereof, or to the use of Table 3 or selected co-ordinates thereof, will be understood to refer to the use of either or both of the A or B chain. Where selected coordinates are used, the selected coordinates preferably will be derived from the same chain.
  • Table 4 provides a model of the human CD44 generated (see below) from the structure of Table 2, and is in a similar format to that Table.
  • Table 5 provides a homology model containing the Link domain of LYVE-1 (residues 29-146).
  • Tables 1 to 5 are set out in internally consistent formats. For example, in Tables 1, 2 and 4 the co-ordinates of the atoms of each amino acid residue are listed such that the backbone nitrogen atom is first, followed by the C-alpha backbone carbon atom, designated CA, followed by side chain residues (designated according to one standard convention) and finally the carbon and oxygen of the protein backbone. In Tables 3 and 5 the carbonyl (C and O) atoms precede the side chain atoms.
  • any suitable standard file format e.g.
  • the co-ordinates of Tables 1 to 5 provide a measure of atomic location in Angstroms, to 3 decimal places.
  • the co-ordinates are a relative set of positions that define a shape in three dimensions, but the skilled person would understand that an entirely different set of coordinates having a different origin and/or axes could define a similar or identical shape.
  • the skilled person would understand that changing the number and/or positions of the sugar or water molecules of Tables 1 to 5 will not generally affect the usefulness of the structure for structure-based analysis of CD44-interacting structures.
  • the Table 1 to 5 co-ordinates are transposed to a different origin and/or axes; the relative atomic positions of the atoms of the structure are varied so that the root mean square deviation from Table 1 to 5 is less than 1.5 A, preferably less than 1.0 A, more preferably less than 0.5 A, and most preferably less than 0.2 A; and/or the number and/or positions of sugar or water molecules is varied.
  • Reference herein to the co-ordinate data of Tables 1 to 5 and the like thus includes the coordinate data in which one or more individual values of the Table are varied in this way.
  • reference to an rmsd of less than 1.5 A will be understood to include reference to the above, smaller, preferred values.
  • Protein structure similarity is routinely expressed and measured by the root mean square deviation (rmsd), i.e. the square root of the arithmetic mean of the squares of the deviations from the mean. This measures the difference in positioning in space between two sets of equivalent atoms after their optimal superposition.
  • the rmsd can be calculated over all atoms, over residue backbone atoms (i.e. the nitrogen-carbon-carbon backbone atoms of the protein amino acid residues), main chain atoms only (i.e. the nitrogen-carbon-oxygen-carbon backbone atoms of the protein amino acid residues), side chain atoms only or more usually over C-alpha atoms only.
  • the rmsd can be calculated over any of these, using any of the methods outlined below. Preferably, the calculation is performed over all the atoms.
  • Programs for determining rmsd include MNYFIT (part of a collection of programs called COMPOSER, Sutcliffe, M.J., Haneef, L 1 Carney, D. and Blundell, T.L. (1987) Protein Engineering, 1 , 377-384), MAPS (Lu, G. An Approach for Multiple Alignment of Protein Structures (1998, in manuscript and on http://bioinfo1.mbfys.lu.se/TOP/maps.html)).
  • Rmsd can also be calculated using programs such as LSQKAB (Collaborative Computational Project 4. The CCP4 Suite: Programs for Protein Crystallography, Acta Crystallographies, D50, (1994), 760-763), QUANTA (Jones et al., Acta Crystallography A47 (1991), 110-119 and commercially available from Accelerys, San Diego, CA), Insight (commercially available from Accelerys, San Diego, CA), Sybyl® (commercially available from Tripos, Inc., St Louis), O (Jones et al., Acta Crystallographica, A47, (1991), 110-119), and other co-ordinate fitting programs.
  • LSQKAB Collaborative Computational Project 4. The CCP4 Suite: Programs for Protein Crystallography, Acta Crystallographies, D50, (1994), 760-763), QUANTA (Jones et al., Acta Crystallography A47 (1991), 110-119 and commercially available from Accelerys, San
  • the user can define the residues in the two proteins that are to be paired for the purpose of the calculation.
  • the atomic co-ordinates can then be superimposed according to this alignment and an rmsd value calculated.
  • the program Sequoia CM. Bruns, I. Hubatsch, M. Ridderstr ⁇ m, B. Mannervik, and J.A. Tainer (1999) Human Glutathione Transferase A4-4 Crystal Structures and Mutagenesis Reveal the Basis of High Catalytic Efficiency with Toxic Lipid Peroxidation Products, Journal of Molecular Biology 288(3): 427-439) performs the alignment of homologous protein sequences, and the superposition of homologous protein atomic co-ordinates.
  • selected co-ordinates means for example at least 5, preferably at least 10, more preferably at least 50 and even more preferably at least 100, for example at least 500 or at least 1000 atoms of the CD44 structure, or other structures referred to herein. Reference herein to selected co-ordinates in all the various applications of the invention described herein is to be understood as including these preferred numbers of such co-ordinates.
  • the selected co-ordinates preferably include at least one atom from the amino acids in the group consisting of Arg45, Tyr46, Cys81 , Arg82, Tyr83, Ile92, Asn98, Ile100, Cys101 , Ala102, Ala103, His105, Tyr109 and Arg155.
  • the residues Arg45 and Arg155 do not appear to make direct ligand contact.
  • the position of Arg45 is significantly shifted and thus appears to have an important role in the ligand-binding process.
  • both residues, and particularly Arg45 may be included in the selected co-ordinates.
  • At least one atom from the above-mentioned group is from Arg45 or Arg155, then at least a second atom of another amino acid is also selected.
  • Reference to the above-mentioned group is to be construed accordingly throughout this disclosure.
  • the selected co-ordinates include at least one atom from at least one of Cys81 , Ile92, Asn98, HeIOO, Cys101 , Ala102 and Ala103 such as at least one atom from Cys81 ,
  • the selected co-ordinates include at least one atom from at least one of Cys81 , Ile92, Asn98, HeIOO 1 Cys101 , Ala102 and Ala103, preferably from at least one of Cys81 , lle100, Cys101 , Ala102 and Ala103, together with at least one atom of a second residue selected from Arg45, Tyr46, Cys81 , Arg82, Tyr83, Ne92, Asn98, HeIOO, Cys101 , Ala102, Ala1 O3, His105, Tyr109 and Arg155, particularly Arg45.
  • the selected co-ordinates when the selected co-ordinates include at least one atom from the group of residues Cys81 , Ile92, Asn98, IleiOO, Cys101 , Ala102 and Ala103, and more preferably from the group of residues Cys81 , IleiOO, Cys101 , Ala102 and Ala103, the selected co-ordinates include at least one atom from at least 2, such as at least 3, more preferably at least 4, even more preferably at least 5 and most preferably all amino acids of these preferred groups. More preferably, the selected co-ordinates comprise at least 10, more preferably 25, more preferably 50 atoms from these groups of residues wherein at least one atom is from each member of the group.
  • the coordinate data of Table 2 or selected coordinates thereof are preferred, particularly wherein said coordinates include one or more from the group of residues Cys81 , Ile92, Asn98, Ile100, Cys101 , AIaI 02 and AIaI 03 together with one or more Arg45 atoms.
  • the selected co-ordinates preferably include at least one atom from the amino acids in the group consisting of Arg41 ,Tyr42, Cys77, Arg78, Tyr79, lle ⁇ , Asn94, Ile96, Cys97, Ala98, Ala99, Asn101 , TyM 05 and Arg150.
  • Arg41 may be included in the selected co-ordinates.
  • Reference to the above-mentioned group is to be construed accordingly throughout this disclosure.
  • the selected co-ordinates include at least one atom from at least one of Cys77, Ile88, Asn94, Ne96, Cys97, Ala98 and Ala99 such as at least one atom from Cys77, Ne96, Cys97, Ala98 and Ala99.
  • the selected co-ordinates include at least one atom from at least one of Cys77, lle ⁇ , Asn94, Ile96, Cys97, Ala98 and Ala99 , preferably from at least one of Cys77, Ile96, Cys97, Ala98 and Ala99, together with at least one atom of a second residue selected from Arg41 ,Tyr42, Cys77, Arg78, Tyr79, lle ⁇ , Asn94, Ile96, Cys97, Ala9 ⁇ , Ala99, Asn101 , TyM 05 and Arg150.
  • the selected co-ordinates include at least one atom from the group of residues Cys77, lle ⁇ , Asn94, Ile96, Cys97, Ala98 and Ala99 , and more preferably from the group of residues Cys77, Ile96, Cys97, Ala98 and Ala99
  • the selected coordinates include at least one atom from at least 2, such as at least 3, more preferably at least 4, even more preferably at least 5 and most preferably all amino acids of these preferred groups. More preferably, the selected co-ordinates comprise at least 10, more preferably 25, more preferably 50 atoms from these groups of residues wherein at least one atom is from each member of the group.
  • selected co-ordinates of Table 3 or 4 includes the above preferred combinations of selected co-ordinates, generally derived, in the case of Table 3, either from the A chain or from the B chain of the structure.
  • the above-mentioned selected co-ordinates are preferred as co-ordinates to be fitted to a ligand structure.
  • the coordinate data of Table 4 or selected coordinates thereof are preferred, particularly wherein said coordinates include one or more from the group of residues Cys77, Ile88, Asn94, Ile96, Cys97, Ala98 and Ala99 together with one or more Arg41 atoms.
  • the co-ordinate when the selected co-ordinates include one or more coordinates of any one of the amino acids set out in Table 6, the co-ordinate is, or includes, the specific atom of column 3 Table 6.
  • the co-ordinate is, or includes, the specific atom of column 3 Table 6.
  • the co-ordinates of any one of the residues set out in Table 6, either singly or in combination with other co-ordinates will be understood, in a preferred aspect, to include the use of the co-ordinates of the specific atom(s) of Table 6, for example of 2 or more, such as 4 or more, for example from 6 to 12 atoms of Table 6.
  • Other co-ordinates of the same residue may also be used at the same time.
  • the selected coordinates include at least one atom from Cys ⁇ , Tyr87, Arg99, Asn103, Lys105, Cys106, Gly107, LysiO ⁇ and Trp116. More preferably the selected coordinates include at least one atom from at least one of Cys ⁇ , Lys105, Cys106, Gly107 and Lys108.
  • the selected co-ordinates include at least one atom from at least one of Cys ⁇ , LysiO ⁇ , Cys106, Gly107 and Lys108, together with at least one atom of a second residue selected from Cys ⁇ , Tyr ⁇ 7, Arg99, Asn103, LysiO ⁇ , Cys106, Gly107, LysiO ⁇ and Trp116.
  • the selected co-ordinates include at least one atom from the group of residues Cys85, Lys105, Cys106, Gly107 and Lys108
  • the selected coordinates include at least one atom from at least one atom from at least 2, such as at least 3, more preferably at least 4 amino acids of this group. More preferably, the selected coordinates comprise at least 10, more preferably 25, more preferably 50 atoms from these groups of residues wherein at least one atom is from each member of the group.
  • selected co-ordinates of Table 5 includes the above preferred combinations of selected co-ordinates.
  • the above-mentioned selected co-ordinates are preferred as co-ordinates to be fitted to a ligand structure.
  • selected co-ordinates of any of Tables 1 to 5 includes the above preferred combinations of selected co-ordinates.
  • the above-mentioned selected co-ordinates are preferred as co-ordinates where at least one of which is to be fitted to a ligand structure.
  • the present invention provides systems, particularly a computer system, the systems containing the atomic co-ordinate data of the of any one of Tables 1 to 5, or selected co-ordinates thereof, in particular the combinations and groups referred to above.
  • the computer system may comprise: (i) a computer-readable data storage medium comprising data storage material encoded with the computer-readable data; (ii) a working memory for storing instructions for processing said computer-readable data; and (iii) a central- processing unit coupled to said working memory and to said computer-readable data storage medium for processing said computer-readable data and thereby generating structures and/or performing rational drug design.
  • the computer system may further comprise a display coupled to said central-processing unit for displaying said structures.
  • Such data is useful for a number of purposes, including the generation of structures to analyse the mechanisms of action of CD44 or LYVE-1 and/or to perform rational drug design of compounds, which interact with CD44 or LYVE-1.
  • the present invention provides computer system containing computer- readable data comprising atomic co-ordinate data of Tables 1 , 2, 4 or 5 optionally varied by a rmsd of less than 1.5A or selected co-ordinates thereof.
  • said selected co-ordinates include at least one atom from the amino acids Arg45, Tyr46, Cys81 , Arg82, Tyr83, Ile92, Asn98, HeIOO 1 Cys101 , Ala102, Ala103, His105, TyM 09 and Arg 155 in the case of Table 1 or 2, at least one atom from the amino acids Arg41 ,Tyr42, Cys77, Arg78, Tyr79, Ile88, Asn94, Ile96, Cys97, Ala98, Ala99, Asn101 , TyM 05 and Arg 150 in the case of Table 4, or at least one atom from the amino acids Cys85, Tyr87, Arg99, Asn103, Lys105, Cys106, Gly107, Lys
  • the invention provides a computer-readable storage medium, comprising a data storage material encoded with computer readable data, wherein the data are defined atomic co-ordinate data of Table 1 or 2 optionally varied by a rmsd of less than 1.5A or selected co-ordinates thereof.
  • said selected co-ordinates include at least one atom from the amino acids Arg45, Tyr46, Cys81 , Arg82, Tyr83, Ile92, Asn98, MeIOO, Cys101 , Ala102, Ala103, His105, Tyr109 and Arg155. More preferred selected co-ordinates are those particular combinations and groups referred to above.
  • the invention provides a computer-readable storage medium, comprising a data storage material encoded with computer readable data, wherein the data are defined atomic co-ordinate data of Table 4 optionally varied by a rmsd of less than 1.5A or selected coordinates thereof.
  • said selected co-ordinates include at least one atom from the amino acids Arg41 ,Tyr42, Cys77, Arg78, Tyr79, Ile88, Asn94, Ile96, Cys97, Ala98, Ala99, Asn101 , Tyr105 and Arg150. More preferred selected co-ordinates are those particular combinations and groups referred to above.
  • the invention provides a computer-readable storage medium, comprising a data storage material encoded with computer readable data, wherein the data are defined atomic co-ordinate data of Table 5 optionally varied by a rmsd of less than 1.5A or selected co- ordinates thereof.
  • said selected co-ordinates include at least one atom from the amino acids Cys85, Tyr87, Arg99, Asn103, Lys105, Cys106, Gly107, Lys108 and Trp116. More preferred selected co-ordinates are those particular combinations and groups referred to above.
  • computer readable media refers to any medium or media, which can be read and accessed directly by a computer. Such media include, but are not limited to: magnetic storage media such as floppy discs, hard disc storage medium and magnetic tape; optical storage media such as optical discs or CD-ROM; electrical storage media such as RAM and ROM; and hybrids of these categories such as magnetic/optical storage media.
  • RASMOL Single et al., TIBS, Vol. 20, (1995), 374
  • TIBS TIBS, Vol. 20, (1995), 374
  • RASMOL is a publicly available computer software package, which allows access and analysis of atomic co-ordinate data for structure determination and/or rational drug design.
  • a computer system refers to the hardware means, software means and data storage means used to analyse the atomic co-ordinate data derived from any one of Tables 1 , 2, 4 or 5 or selected co-ordinates thereof.
  • the minimum hardware means of the computer- based systems of the present invention comprises a central processing unit (CPU), input means, output means and data storage means. Desirably a monitor is provided to visualize structure data.
  • the data storage means may be RAM or means for accessing computer readable media of the invention. Examples of such systems are microcomputer workstations available from Silicon Graphics Incorporated and Sun Microsystems running Unix based or Windows XP operating systems.
  • the invention also provides a computer-readable data storage medium comprising a data storage material encoded with a first set of computer-readable data comprising any one of
  • a further aspect of the invention provides a method of providing data for generating structures and/or performing rational drug redesign with CD44, CD44 homologues or analogues, complexes of CD44 with a compound, or complexes of CD44 homologues or analogues with compounds, the method comprising:
  • said selected co-ordinates preferably include at least one atom from the amino acids Arg45, Tyr46, Cys81 , Arg82, Tyr83, Ile92, Asn98, IleiOO, Cys101 , Ala102, Ala103, His105, Tyr109 and Arg155 and in the case of Table 3 or 4 said selected coordinates include at least one atom from the amino acids Arg41 ,Tyr42, Cys77, Arg78, Tyr79, Ne88,
  • the invention also provides a method of providing data for generating structures and/or performing rational drug redesign with LYVE-1 , LYVE-1 homologues or analogues, complexes of LYVE-1 with a compound, or complexes of LYVE-1 homologues or analogues with compounds, the method comprising:
  • the selected co-ordinates preferably include at least one atom from the amino acids Cys85, Tyr87, Arg99, Asn103, Lys105, Cys106, Gly107, Lys108 and Trp116.
  • the particular combinations and groups of selected co-ordinates referred to above are preferred.
  • the remote device may comprise e.g. a computer system or computer readable media of one of the previous aspects of the invention.
  • the device may be in a different country or jurisdiction from where the computer-readable data is received.
  • the communication may be via the internet, intranet, e-mail etc, transmitted through wires or by wireless means such as by terrestrial radio or by satellite.
  • the communication will be electronic in nature, but some or all of the communication pathway may be optical, for example, over optical fibres.
  • the data received may then be used in a computer-based method for the analysis of the interaction of a ligand with CD44 or LYVE-1 , said method as being further defined herein.
  • the data of the present invention allows the provision of further Link module domain-containing protein structures which may be used in the same manner as the murine CD44 structure described herein.
  • the identification of novel HA-binding residues allows the existing structure of human CD44 (Table 3) to be analysed and used for the development of ligands as described herein.
  • the invention also allows the present data has provided a model of LYVE-1 in which the HA-binding pocket may be determined by reference to the data provided herein.
  • the small structural core to the CD44-HA interaction increases the chances of obtaining a low molecular weight antagonist.
  • the deviation of the bound HA oligosaccharide from its preferred conformation might be exploited in inhibitor design by selecting chemical entities that already have a conformational preference towards the constrained state seen in the HA-CD44 complex.
  • the structure of the CD44-HA complex disclosed herein will allow the design of a pharmacophore for in silico screening of chemical libraries to obtain small molecule inhibitors with the potential for therapy, e.g. for anti-inflammatory or anti-neoplastic therapy.
  • determination of the three-dimensional structure of CD44 provides important information about the HA-binding site of CD44. This information may then be used for rational design and modification of CD44 substrates and inhibitors, e.g. by computational techniques which identify possible binding ligands for the binding sites, by enabling linked- fragment approaches to drug design, and by enabling the identification and location of bound ligands using X-ray crystallographic analysis. These techniques are discussed in more detail below.
  • CD44 structure in silico may be equally applied to both the CD44 structure from the data of Table 1 or 2, or selected co- ordinates thereof and the models of target Link module domain-containing proteins obtained by other aspects of the invention.
  • a conformation of a protein by the method described above such a conformation may be used in a computer-based method of rational drug design as described herein.
  • the availability of the structure of the CD44 structure will allow the generation of highly predictive pharmacophore models for virtual library screening or compound design.
  • the invention provides a computer-based method for the analysis of the interaction of a ligand with CD44, which comprises: providing the CD44 structure of Table 1 or 2 optionally varied by a rmsd of less than 1 ,5A or selected co-ordinates thereof; providing a ligand structure to be fitted to said CD44 structure or selected co-ordinates thereof; and fitting the ligand structure to said CD44 structure, wherein said ligand structure is fitted to at least one atom from the amino acids Arg45, Tyr46, Cys81 , Arg82, Tyr83, Ile92, Asn98, IleiOO, Cys101 , Ala102, Ala103, His105, Tyr109 and Arg155. More preferred selected coordinates are those particular combinations and groups referred to above.
  • the invention also provides a computer-based method for the analysis of the interaction of a ligand with CD44, which comprises: providing the CD44 structure of Table 3 or 4 optionally varied by a rmsd of less than 1.5A or selected co-ordinates thereof; providing a ligand structure to be fitted to said CD44 structure or selected co-ordinates thereof; and fitting the ligand structure to said CD44 structure, wherein said ligand structure is fitted to at least one atom from the amino acids Arg41 ,Tyr42, Cys77, Arg78, Tyr79, Ile88, Asn94, Iie96, Cys97, Ala98, Ala99, Asn101, Tyr105 and Arg150. More preferred selected co-ordinates are those particular combinations and groups referred to above.
  • the invention further provides a computer-based method for the analysis of the interaction of a ligand with LYVE-1 , which comprises: providing the LYVE-1 structure of Table 5 optionally varied by a rmsd of less than 1.5A or selected co-ordinates thereof; providing a ligand structure to be fitted to said LYVE-1 structure or selected coordinates thereof; and fitting the ligand structure to said LYVE-1 structure, wherein said ligand structure is fitted to at least one atom from the amino acids Cys85, Tyr87, Arg99, Asn103, Lys105, Cys106, GIy 107, Lys108 and Trp116. More preferred selected co-ordinates are those particular combinations and groups referred to above.
  • the ligand structure may be modelled in three dimensions using commercially available software for this purpose or, if its crystal structure is available, the co- ordinates of the structure may be used to provide a representation of the ligand for fitting to a CD44 structure of the invention.
  • the HA binding pocket of CD44 molecules are of a size which can accommodate more than one ligand.
  • the findings of the present invention may be used to examine or predict the interaction of two or more separate molecular structures within the CD44 binding pocket of the invention.
  • the invention provides a computer-based method for the analysis of the interaction of two ligand structures within a CD44 or LYVE-1 binding pocket structure, which comprises: providing the structure of any one of Tables 1 to 5 or selected co-ordinates thereof; providing a first ligand structure; fitting the first ligand structure to said CD44 or LYVE-1 structure; providing a second ligand structure; and fitting the second ligand structure to a different part said CD44 or LYVE-1 structure.
  • the method of analysis further comprises providing a third ligand structure and also fitting that structure to the CD44 or LYVE-1 structure.
  • further ligand structures may be provided and fitted in the same way.
  • a person of skill in the art may seek to use molecular modelling to determine to what extent the structures interact with each other (e.g. by hydrogen bonding, other non-covalent interactions, or by reaction to provide a covalent bond between parts of the structures) or the interaction of one structure with CD44 or LYVE-1 is altered by the presence of another structure.
  • the person of skill in the art may use in silico modelling methods to alter one or more of the structures in order to design new structures which interact in different ways with CD44 or LYVE-1.
  • Newly designed structures may be synthesised and their interaction with CD44 or LYVE-1 may be determined or predicted as to how the newly designed structure binds to CD44 or LYVE-1. This process may be iterated so as to further alter the interaction between it and the CD44 or LYVE-1.
  • fitting it is meant determining by automatic, or semi-automatic means, interactions between at least one atom of a molecular structure and at least one atom of a CD44 or LYVE-1 structure of the invention, and calculating the extent to which such an interaction is stable. Interactions include attraction and repulsion, brought about by H-bonding, charge, steric considerations and the like. Various computer-based methods for fitting are described further herein.
  • the CD44 or LYVE-1 co-ordinates to be fitted preferably include the selected co-ordinates which have been identified as interacting with HA.
  • a ligand or ligands with CD44 or LYVE-1 can be examined through the use of computer modelling using a docking program such as GOLD (Jones et al., J. MoI. Biol., 245, 43-53 (1995), Jones et al., J. MoI. Biol., 267, 727-748 (1997)), GRAMM (Vakser, I.A., Proteins , Suppl., 1 :226-230 (1997)), DOCK (Kuntz et al, J.Mol.Biol. 1982 , 161, 269-288, Makino et al, J.Comput.Chem.
  • GOLD Jones et al., J. MoI. Biol., 245, 43-53 (1995), Jones et al., J. MoI. Biol., 267, 727-748 (1997)
  • GRAMM Vakser, I.A., Proteins , Suppl., 1 :226-230 (1997))
  • Computer programs can be employed to estimate the attraction, repulsion, and steric hindrance of the two binding partners (i.e. a CD44 or LYVE-1 structure and a ligand).
  • a ligand may be formed by linking the respective small ligands into a larger ligand, which maintains the relative positions and orientations of the respective ligands at the active sites.
  • the larger ligand may be formed as a real molecule or by computer modelling.
  • molecular structures which may be fitted to the CD44 or LYVE-1 structure of the invention, include compounds under development as potential pharmaceutical agents.
  • the agents may be useful as anti-inflammatory drugs or for other indications in which CD44 or LYVE-1 -binding to agonize or antagonize the protein activity may be beneficial.
  • Ligands which may be used in the present invention, will usually be compounds under development for pharmaceutical use. Generally such compounds will be organic molecules, which are typically from about 100 to 2000 Da, more preferably from about 100 to 1000 Da in molecular weight. Such compounds include peptides and derivatives thereof, steroids, anti- inflammatory drugs, anti-cancer agents, anti-bacterial or antiviral agents, neurological agents and the like. In principle, any compound under development in the field of pharmacy can be used in the present invention in order to facilitate its development or to allow further rational drug design to improve its properties.
  • a ligand which may be used is a pharmacophore structure comprising three positions arranged to correspond geometrically to the positions of atoms 1188, 1193 and 1196 of Table 1 or 2, or the corresponding positions in Tables 3 or 4.
  • the atoms of the pharmacophore may also correspond chemically to the atoms at these positions, i.e. may be O, N and C respectively, though other atoms with similar chemical and/or physical properties may also be used.
  • correspond geometrically it is meant that the pharmacophore contains three atoms whose relationship in three-dimensional space (i.e. distance and angles between the atoms) is substantially the same as the distances and angles between the positions of the above- mentioned atoms.
  • the structure of the ligand may be modelled in order to determine residues of CD44 or LYVE-1 which interact with the agent.
  • the present invention provides a process for predicting potential pharmaceutical compounds with the potential to bind to CD44 or LYVE-1.
  • This may be performed by designing ligands in accordance with the invention as described herein, then iterating the process, for example by: modifying the ligand structure to reduce or increase at least one interaction with the CD44 or LYVE-1 structure; and optionally further obtaining or synthesising a compound which has said ligand structure; forming a complex of a CD44 or LYVE-1 protein and said compound; and analysing said complex by X-ray crystallography to determine the ability of said compound to interact with the CD44 or LYVE-1 protein.
  • Modification will be those conventional in the art known to the skilled medicinal chemist, and will include, for example, substitutions or removal of groups containing residues which interact with the amino acid side chain groups of a CD44 or LYVE-1 structure of the invention.
  • the replacements may include the addition or removal of groups in order to decrease or increase the charge of a group in a test compound, the replacement of a charge group with a group of the opposite charge, or the replacement of a hydrophobic group with a hydrophilic group or vice versa. It will be understood that these are only examples of the type of substitutions considered by medicinal chemists in the development of new pharmaceutical compounds and other modifications may be made, depending upon the nature of the starting compound and its activity.
  • the invention further includes the step of synthesizing the modified compound and testing it in a in vivo or in vitro biological system in order to determine its activity and/or the rate at which it is metabolised.
  • the binding of one or more molecular fragments can be determined in the protein binding pocket by X-ray crystallography.
  • Molecular fragments are typically compounds with a molecular weight between 100 and 200 Da (Carr et al, Drug Discov Today. 2002 May 1;7(9):522- 7). This can then provide a starting point for medicinal chemistry to optimise the interactions using a structure-based approach.
  • the fragments can be combined onto a template or used as the starting point for 'growing out' an inhibitor into other pockets of the protein (Blundell et al, Nat Rev Drug Discov. 2002 Jan;1(1):45-54).
  • the fragments can be positioned in the HA binding pocket of the CD44 or LYVE-1 and then 'grown' to fill the space available, exploring the electrostatic, van der Waals or hydrogen-bonding interactions that are involved in molecular recognition.
  • the potency of the original weakly binding fragment thus can be rapidly improved using iterative structure-based chemical synthesis.
  • one ligand may be fitted to another domain of the CD44 or LYVE-1 protein, such that this approach is used to develop a ligand which binds both to the HA-domain and to a separate domain of the protein.
  • the compound may be synthesized and tested in a biological system for its activity. This can be used to guide the further growing out of the fragment.
  • a linked fragment approach may be based upon attempting to link the two fragments directly, or growing one or both fragments in the manner described above in order to obtain a larger, linked structure, which may have the desired properties.
  • binding site of two or more ligands may be connected to form a potential lead compound that can be further refined using e.g. the iterative technique of Greer et al.
  • Greer et al. For a virtual linked-fragment approach see Verlinde et al., J. of Computer-Aided Molecular Design, 6, (1992), 131-147, and for NMR and X-ray approaches see Shuker et al., Science, 274, (1996), 1531-1534 and Stout et al., Structure, 6, (1998), 839-848.
  • the invention further includes the step of synthesizing the compound and testing it in an in vivo or in vitro biological system in order to determine its activity, e.g. its ability to act as an agonist or antagonist of CD44 or LYVE-1.
  • Compounds which modulate the activity of CD44 have potential use in the treatment of inflammatory diseases, such as arthritis, autoimmune diabetes and asthma/pulmonary eosinophilia.
  • LYVE-1 may therefore prove to be an attractive target for the development of small molecule HA-blocking drugs for potential therapy of inflammatory disease involving the lymphatic network.
  • the invention includes a compound, which is identified by the methods of the invention described above.
  • a compound may be manufactured and/or used in the preparation, i.e. manufacture or formulation, of a composition such as a medicament, pharmaceutical composition or drug. These may be administered to individuals.
  • the present invention extends in various aspects not only to a compound as provided by the invention, but also a pharmaceutical composition, medicament, drug or other composition comprising such a compound.
  • the compositions may be used, for treatment (which may include preventative treatment) of disease such as inflammation.
  • a treatment may comprise administration of such a composition to a patient, e.g. for treatment of disease; the use of such an inhibitor in the manufacture of a composition for administration, e.g. for treatment of disease; and a method of making a pharmaceutical composition comprising admixing such an inhibitor with a pharmaceutically acceptable excipient, vehicle or carrier, and optionally other ingredients.
  • a further aspect of the present invention provides a method for preparing a medicament, pharmaceutical composition or drug, the method comprising: (a) identifying or modifying a compound by a method of any one of the other aspects of the invention disclosed herein; (b) optimising the structure of the molecule; and (c) preparing a medicament, pharmaceutical composition or drug containing the optimised compound.
  • optimisedising the structure we mean e.g. adding molecular scaffolding, adding or varying functional groups, or connecting the molecule with other molecules (e.g. using a fragment linking approach) such that the chemical structure of the modulator molecule is changed while its original modulating functionality is maintained or enhanced.
  • Such optimisation is regularly undertaken during drug development programmes to e.g. enhance potency, promote pharmacological acceptability, increase chemical stability etc. of lead compounds.
  • Modification will be those conventional in the art known to the skilled medicinal chemist, and will include, for example, substitutions or removal of groups containing residues which interact with the amino acid side chain groups of a CD44 or LYVE-1 structure of the invention.
  • the replacements may include the addition or removal of groups in order to decrease or increase the charge of a group in a test compound, the replacement of a charge group with a group of the opposite charge, or the replacement of a hydrophobic group with a hydrophilic group or vice versa. It will be understood that these are only examples of the type of substitutions considered by medicinal chemists in the development of new pharmaceutical compounds and other modifications may be made, depending upon the nature of the starting compound and its activity.
  • compositions may be formulated for any suitable route and means of administration.
  • Pharmaceutically acceptable carriers or diluents include those used in formulations suitable for oral, rectal, nasal, topical (including buccal and sublingual), vaginal or parenteral (including subcutaneous, intramuscular, intravenous, intradermal, intrathecal and epidural) administration.
  • the formulations may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy.
  • conventional non-toxic solid carriers include, for example, pharmaceutical grades of mannitol, lactose, cellulose, cellulose derivatives, starch, magnesium stearate, sodium saccharin, talcum, glucose, sucrose, magnesium carbonate, and the like may be used.
  • Liquid pharmaceutically administrable compositions can, for example, be prepared by dissolving, dispersing, etc, an active compound as defined above and optional pharmaceutical adjuvants in a carrier, such as, for example, water, saline aqueous dextrose, glycerol, ethanol, and the like, to thereby form a solution or suspension.
  • the pharmaceutical composition to be administered may also contain minor amounts of non-toxic auxiliary substances such as wetting or emulsifying agents, pH buffering agents and the like, for example, sodium acetate, sorbitan monolaurate, triethanolamine sodium acetate, sorbitan monolaurate, triethanolamine oleate, etc.
  • auxiliary substances such as wetting or emulsifying agents, pH buffering agents and the like, for example, sodium acetate, sorbitan monolaurate, triethanolamine sodium acetate, sorbitan monolaurate, triethanolamine oleate, etc.
  • wetting or emulsifying agents for example, sodium acetate, sorbitan monolaurate, triethanolamine sodium acetate, sorbitan monolaurate, triethanolamine oleate, etc.
  • Actual methods of preparing such dosage forms are known, or will be apparent, to those skilled in this art; for example, see Remington's
  • HA oligosaccharides of defined length were produced as described in Mahoney et al. (23).
  • Biotinylated-HA (b-HA) was prepared from Genzyme medical grade HA (-1.2 MDa) as described in Mahoney et al., (23).
  • the monoclonal antibody IM7 was obtained from Pharmingen (Oxford, UK).
  • Two further antibodies specific for murine CD44, IRAW B14 and KM81 were obtained from Dr. Helen Yarwood (Imperial College, London, UK) and Dr Jayne Lesley (SaIk Institute, San Diego, USA).
  • a full-length cDNA clone for mouse CD44 (the haemopoetic or standard form) was obtained from Dr. Helen Yarwood and sub- cloned into the mammalian expression vector pRcCMV (Invitrogen, Paisley, UK).
  • a segment of mouse CD44 cDNA encoding the HABD was prepared by PCR amplification using the forward primer GGAATTCtcatgaATCAGATCGATTTGAATGTAACCTGCCGC (SEQ ID NO:4) containing a Bsp HI site (lowercase) and the reverse primer
  • GCggatccTCAATCGATGTCTTCTTGGTGTGTTCTATAC (SEQ ID NO:5) containing a Bam HI site for ligation into the expression vector pET19b (Novagen, Nottingham, UK).
  • the polypeptide, CD44 25"174 was equivalent to the first 152 residues of the mature N-terminus of mouse CD44 with the first two residues H 23 Q being replaced by MN. N-terminal sequencing indicated that the translation initiating methionine was retained in the E. coli expressed protein.
  • the HABD was expressed and purified to homogeneity as described previously (24).
  • the HABD construct for human CD44 was prepared and expressed according to the method described in Banerji et al 1998 (24). 15 N labelling of human CD44 20'178 was carried out as described in Teriete et al 2004 (19). Mutated human CD44 constructs were prepared as described below and were expressed and labelled in the same manner as for the wild-type.
  • the protein/HA sample was mixed 1 :1 with a well solution containing 25% (w/v) PEG 3350 and 10OmM NaCI in HEPES buffer pH 7.
  • the protein/HA sample was mixed with 25% PEG 3350 and 10OmM NaCI in 10OmM Hepes buffer pH8. Co-crystals grew over a period of two to eight weeks at room temperature. NMR spectroscopy
  • Samples for NMR were prepared from lyophilized hCD44 20"178 (0.3 mM: wild-type, R41A, R150A, R154A; 0.15 mM: K158A and R162A) in 10% (v/v) D 2 O (containing 0.02% [w/v] Na- azide) and adjusted to pH 6.5.
  • 1 H 1 15 N-HSQC spectra were acquired at 500 MHz in the absence and presence of HA 6 or HAi 0 at oligosaccharide to protein stoichiometries of 0:1 , 1 :1 , 2:1 , 3:1 and 4:1 as described before for the wild-type HABD (19).
  • the single site mutants K38A, R41A, R150A, R154A, K158A and R162A of human CD44 20"178 were made using the Transformer Site-Directed Mutagenesis Kit (Clontech) and expressed in E. coli as described previously for the wild-type protein (24).
  • the triple mutant DGT151-153AAA was constructed for analysis of HA binding by surface plasmon resonance. Mutagenesis was carried out according to the QuickChangeTM mutatgenesis procedure using the forward primer GGACCAATTACCATAACTATTGTTAACCGTGCTGCCGCCCGCTATGTCCAGAAAGG (SEQ ID NO: 10) and the reverse primer
  • Recombinant pRcCMV plasmids designed for cell surface expression of full-length wild-type mouse CD44 and the mutants described above were transiently transfected into the CD44 negative human 293T fibroblast cell line using calcium phosphate precipitation.
  • Transfected cells were stained with FITC-HA and the mouse CD44 specific antibody IM7.
  • Transfectants were stained with two additional antibodies KM81 and IRAWB14 (also specific for mouse CD44) to assess expression levels of the mutated CD44 compared to the wild-type.
  • SPR Surface plasmon resonance
  • binding analyses were performed using various concentrations of wild-type (0-169 ⁇ M) or mutant human CD44 20'178 (0-197 ⁇ M R150A, 0-99 ⁇ M R154A, 0-59 ⁇ M K168A, 0-45 ⁇ M R162A), where 40 ⁇ l of the protein solution was flowed over the sensor chip surface.
  • the analyte was simultaneously passed over a blank flow cell (i.e., no with b-HA coupled) and this reference sensogram was subtracted from the data derived from the experimental flow cell. From these adjusted data, the maximum equilibrium binding was determined 125 s following the beginning of the injection.
  • Oligosaccharide solutions (ranging from 1.93 to 8.47 ⁇ M, where the concentrations were determined on the basis of a control experiment with Link_TSG6) were added in 28 x 5- ⁇ l injections to protein at 0.176-0.404 mM; protein concentrations were determined by amino acid analysis (23). There was a spacing of 300 s between injections and a reference power of 15 ⁇ cal/s was used for all of the titrations. Data were fitted to a one-site model by non-linear least squares regression with the Origin software package, after subtracting heats resulting from the addition of oligomer into buffer alone, as described previously (23, 27).
  • the structure was modelled by mutating all of the sequence differences in the HA binding domain of mouse CD44 to their human equivalents, and choosing the side chain rotamer that gave the least possible clashes.
  • the loop region spanning residues 108-112 (which contains the deletion) was adopted from the human apoprotein structure, and the structure was energy minimised to relieve any strain thus introduced.
  • the bound HA oligomer can be seen to occupy a discrete binding patch in a groove that extends from the side of the hook-like loop between ⁇ strands 4 and 5 to the channel that connects ⁇ 3, ⁇ 4 and ⁇ 5 (previously referred to as the SII sheet) with the rest of the Link module.
  • the core of sugar binding is therefore centred on a relatively small region of the HA octamer, involving primarily residues GlcUA5 through GlcNAc ⁇ . This is reflected in the large temperature factor measured for GlcNAc2 (42.3 A 2 ) and the much lower values for GlcUA5 through GlcNAc ⁇ (14.0 - 17.5 A 2 ).
  • the relatively short footprint is apparent also from considerations of the conformational flexibility of bound HA.
  • HA bound to protein can be expected to experience conformational constraints, as a consequence of sugar-protein interactions and intramolecular hydrogen bonds between neighbouring sugar residues. It was also observed that residues GlcNac 2 through GIcNAc 4 can be superimposed precisely on the preferred conformation of the sodium salt of an HA 8-mer. However, superimposition of sugars GlcNac 4- GIcNAc 8 on the HA 8mer shows that this portion of the chain is distorted, confirming the conformational constraint on these residues imposed by interaction with the binding cleft on CD44.
  • a conformational flip of the main-chain around residue G44 results in a side- chain rearrangement that brings the key residue R45 into contact with HA in crystal B, thereby displacing R82 from interacting with HA and producing a subtle shift in E41.
  • the effect of this conformational change which may well be induced by ligand-binding, would be to increase the binding affinity for HA and crystal form B may thus be the conformation of the receptor that predominates in the active form of the receptor at the cell surface.
  • a novel feature of this interaction is the insertion of the methyl group of the acetyl moiety into a "pocket" which is lined by a number of (polar) main chain carbonyls in addition to the hydrophobic sidechains of Y83, and I92, each of which engage in H bonds with the sugar ring.
  • the predominance of H bonds in the binding groove is also apparent for the remaining sugar interactions.
  • GlcNAc4 is co-ordinated by H- bonds to N98 and C101
  • GlcUA7 is H bonded to Y83, 1100, C101 , A102, and A103
  • GlcNac ⁇ H-bonded to A103 C ⁇ -C4/ C ⁇ -04
  • H 105 N ⁇ 2-O6
  • Y109 ring OH-C6/ring OH to O6/C ⁇ -O6/
  • the binding of the hCD44 20'178 mutants to polymeric HA was also investigated by SPR, where b-HA was coupled to a streptavidin-coated sensor chip. Preliminary experiments were conducted where the wild-type and mutant proteins were flowed over the chip surface at 0-12 ⁇ M. This demonstrated that a reasonable binding signal could be detected in all cases apart for the R41A mutant, which at 10 ⁇ M only gave 17 response units (RU) compared to 438 for the wild-type protein (i.e., after subtraction of the reference sensogram). This is consistent with the low affinity of R41 A for HA seen by microtitre plate assay and NMR; no further SPR experiments were carried out for this mutant.
  • CD44 hyaluronan-binding site revealed by the present crystal structure indicates no obvious contribution from residues within the C and N-terminal Link extensions, formed from the four supplementary ⁇ strands 0, 7, 8 and 9 that are essential for correct folding and function of the HA-binding domain (19, 29).
  • a group of basic amino acids located within and around ⁇ 9 had previously shown to be important for HA- binding to human CD44 through alanine mutagenesis (21 ; 22).
  • cDNAs encoding full-length wild-type receptor and site-directed mutants were transfected into the 293T cell line and the capacity to bind FI-HA assessed as a function of surface expression and authentic folding using the murine-reactive mAb IM7 and two conformation selective mAbs IRAWB14 and KM81.
  • Mutation of either K42, R159, K163 or R167 to alanine had little if any effect on the ability of the receptor to bind FI-HA, measured at either high or low CD44 surface densities.
  • mutation of R155 reduced HA binding significantly compared to wild-type CD44, although not to the same extent as mutation of either R45 or A103.
  • R150A R155A
  • R154A R159A
  • K158A K163A
  • R162A R167A
  • the CD44-HA complex packs in the crystal lattice in such a way that the bound oligosaccharide units of neighbouring CD44 molecules resemble parallel segments of a single co-linear high molecular weight HA polymer.
  • This pattern brings the individual CD44 molecules in sufficiently close proximity to suggest a protein:protein interface between adjacent molecules involving the loops between ⁇ 8 - ⁇ 9 on one molecule and the ⁇ 5- ⁇ 6 loop of its nearest neighbour.
  • contacts in a crystal lattice may not necessarily reflect such contacts in solution. Nevertheless, it is believed to speculate they might resemble interactions that occur transiently on the surface of a cell engaging in multivalent binding to an HA polymer, as they could provide an element of co-operativity to the binding of HA.
  • CD44-HA interaction Certain unusual features of the CD44-HA interaction revealed from the crystal structure of the complex, namely the short sugar footprint, and the preponderance of H-bonds to the CD44 binding groove, affect both the validation of CD44 as a rational drug design target, and the approach which would be taken to design inhibitors.
  • the small structural core to the CD44-HA interaction that is centred on the GIcNAd -GlcUA4 tetrasaccharide increases the chances of obtaining a small molecule antagonist.
  • the largely H-bonded nature of the CD44-HA interaction means that improvement of the binding of a lead compound can be pursued by targetting polar and hydrophobic interactions instead of charged interactions. Interactions that are dominated by ionic interactions represent poor targets for therapeutic interaction, as complementary charges in candidate drugs often make for poor bioavailability.
  • HA receptor LYVE-1 A potentially similar HA-bindinq surface in the lymphatic endothelial hyaluronan receptor LYVE-1.
  • the HA receptor LYVE-1 is expressed almost exclusively in endothelial cells of the lymphatic vasculature, a network that interweaves with but is developmentally distinct from the blood vasculature (5, 16).
  • LYVE-1 cDNA was identified from homology searches of a commercial database (Human Genome Sciences/TIGR) and expressed the protein as a soluble Fc fusion partner (5).
  • Subsequent analysis of ligand-binding and tissue expression using specific polyclonal and monoclonal antibodies confirmed LYVE-1 as an authentic receptor for HA with an apparent binding affinity and specificity for HA that is broadly similar to that of CD44.
  • LYVE-1 is confined to lymphatic endothelium, and is absent from blood vascular endothelium and virtually all other cell types with the exception of liver and spleen sinusoidal endothelia and some populations of tissue macrophages (5, 16, 19).
  • the receptor has been shown to bind HA in a saturable manner that may be blocked specifically by excess free HA, or by the monoclonal antibody 3A that has been generated against rhLYVE-1 in mice ((20) and see below for details of epitope analysis).
  • the LYVE-1 structure model exhibits a core Link domain made up of two ⁇ -sheets ( ⁇ 1 ,2, 6 and ⁇ 3,4,5) flanked by two short a helices ( ⁇ 1, 2).
  • this core structure is extended by additional ⁇ -strands ( ⁇ O and ⁇ 7) that are linked by a third disulphide bridge contributed from sequences N and C- terminal to the consensus Link module.
  • the LYVE-1 HA-binding domain appears to lack the equivalent of ⁇ -strands 8 and 9 and is therefore likely to be a more compact structure.
  • a potential HA-binding face has been identified on the surface of the LYVE-1 structure by plotting the positions of amino acids defined as essential (Tyr87, Ile97 and Arg99) or important for ligand-binding (including Asn103, Lys105, Lys108 and Trp116) through site- directed mutagenesis studies.
  • Tyr87, Arg99 and Trp116 have been identified as part of the epitope for the HA-blocking mAb 3A. Overall these residues define a coherent patch that is similar in nature and location to the ligand binding face identified in the mCD44:HA crystal complex.
  • the 115 compounds are assayed initially for their ability to block binding of biotinylated high molecular weight HA to purified immobilized CD44 in an established 96 well plate ELISA
  • TNF ⁇ induces functionally active hyaluronan-adhesive CD44 by activating silidase through p38 mitogen-activated protein kinase in lipopolysaccharide-stimulated human monocytic cells. J. Biol. Chem. 278:37275-37287.
  • LYVE-1 a new homologue of the CD44 glycoprotein, is a lymph-specific receptor for hyaluronan. J Cell Biol 144:789-801.
  • ATOM 363 CA LYS A 71 -9.539 -10.333 1.419 1.00 5.25
  • ATOM 408 CA GLY A 77 -2.344 -13.629 -5.063 1.00 6.32
  • ATOM 456 CA TYR A 83 2.640 -6.480 9.595 1.00 4.44
  • ATOM 552 CA ILE A 95 -1.410 -17.547 1..145 .00 4.82
  • ATOM 570 CA PRO A 97 1.925 -21.200 5.564 00 6.56

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  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Peptides Or Proteins (AREA)

Abstract

L'invention concerne la structure cristalline de la molécule de la protéine CD44. Cette structure est présentée dans les tableaux de 1 à 4. On peut utiliser ladite structure pour modéliser l'interaction de composés, tels que des produits pharmaceutiques, avec la protéine et déterminer la structure des molécules associées.
PCT/GB2006/003734 2005-10-06 2006-10-06 Structure cristalline de cd44 et son utilisation WO2007039761A2 (fr)

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Application Number Priority Date Filing Date Title
GBGB0520365.8A GB0520365D0 (en) 2005-10-06 2005-10-06 Crystal structure of CD44 and its use
GB0520365.8 2005-10-06

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WO2007039761A2 true WO2007039761A2 (fr) 2007-04-12
WO2007039761A3 WO2007039761A3 (fr) 2007-06-21

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20110053864A1 (en) * 2009-03-06 2011-03-03 Angstrom Pharmaceuticals, Inc. Compositions and methods for modulation of cell migration
US8697629B2 (en) 2010-03-05 2014-04-15 Angstrom Pharmaceuticals, Inc. Modulation of intracellular signaling
WO2021233962A1 (fr) 2020-05-19 2021-11-25 Institut Curie Méthodes de diagnostic et de traitement du syndrome de libération de cytokines

Citations (1)

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Publication number Priority date Publication date Assignee Title
WO2003094843A2 (fr) * 2002-05-10 2003-11-20 Incyte Corporation Proteines d'adhesion cellulaire et a matrice extracellulaire

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Publication number Priority date Publication date Assignee Title
WO2003094843A2 (fr) * 2002-05-10 2003-11-20 Incyte Corporation Proteines d'adhesion cellulaire et a matrice extracellulaire

Non-Patent Citations (6)

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Title
BANERJI SUNEALE ET AL: "LYVE-1, a new homologue of the CD44 glycoprotein, is a lymph-specific receptor for hyaluronan" THE JOURNAL OF CELL BIOLOGY, ROCKEFELLER UNIVERSITY PRESS, US, vol. 144, no. 4, 22 February 1999 (1999-02-22), pages 789-801, XP002159438 ISSN: 0021-9525 *
BLUNDELL T L ET AL: "HIGH-THROUGHPUT CRYSTALLOGRAPHY FOR LEAD DISCOVERY IN DRUG DESIGN" NATURE REVIEWS. DRUG DISCOVERY, NATURE PUBLISHING GROUP, BASINGSTOKE, GB, vol. 1, no. 1, January 2002 (2002-01), pages 45-54, XP009023187 ISSN: 1474-1784 *
HUMPHRIES M J ET AL: "The structure of cell-adhesion molecules." TRENDS IN CELL BIOLOGY FEB 1998, vol. 8, no. 2, February 1998 (1998-02), pages 78-83, XP009079851 ISSN: 0962-8924 *
JACKSON DAVID GERALD ET AL: "Structure of the inflammatory homing receptor CD44 complexed with its pericellular matrix ligand hyaluronan." FASEB JOURNAL, vol. 20, no. 5, Part 2, March 2006 (2006-03), page A915, XP009079901 & EXPERIMENTAL BIOLOGY 2006 MEETING; SAN FRANCISCO, CA, USA; APRIL 01 05, 2006 ISSN: 0892-6638 *
TAKEDA MITSUHIRO ET AL: "Ligand-induced structural changes of the CD44 hyaluronan-binding domain revealed by NMR." THE JOURNAL OF BIOLOGICAL CHEMISTRY 29 DEC 2006, vol. 281, no. 52, 29 December 2006 (2006-12-29), pages 40089-40095, XP002422616 ISSN: 0021-9258 *
TERIETE PETER ET AL: "Structure of the regulatory hyaluronan binding domain in the inflammatory leukocyte homing receptor CD44." MOLECULAR CELL 27 FEB 2004, vol. 13, no. 4, 27 February 2004 (2004-02-27), pages 483-496, XP002422615 ISSN: 1097-2765 cited in the application *

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20110053864A1 (en) * 2009-03-06 2011-03-03 Angstrom Pharmaceuticals, Inc. Compositions and methods for modulation of cell migration
EP2403518A2 (fr) * 2009-03-06 2012-01-11 Angstrom Pharmaceuticals, Inc. Compositions et procédés de modulation de la migration cellulaire
US8313914B2 (en) * 2009-03-06 2012-11-20 Angstrom Pharmaceuticals, Inc. Compositions and methods for modulation of cell migration
EP2403518A4 (fr) * 2009-03-06 2013-01-23 Angstrom Pharmaceuticals Inc Compositions et procédés de modulation de la migration cellulaire
US8697629B2 (en) 2010-03-05 2014-04-15 Angstrom Pharmaceuticals, Inc. Modulation of intracellular signaling
WO2021233962A1 (fr) 2020-05-19 2021-11-25 Institut Curie Méthodes de diagnostic et de traitement du syndrome de libération de cytokines
EP4424711A2 (fr) 2020-05-19 2024-09-04 Institut Curie Procedes de diagnostic et de traitement du syndrome de liberation de cytokines

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GB0520365D0 (en) 2005-11-16
WO2007039761A3 (fr) 2007-06-21

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