WO2012162856A1 - Cristal de récepteur de cytokine - Google Patents

Cristal de récepteur de cytokine Download PDF

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WO2012162856A1
WO2012162856A1 PCT/CN2011/000926 CN2011000926W WO2012162856A1 WO 2012162856 A1 WO2012162856 A1 WO 2012162856A1 CN 2011000926 W CN2011000926 W CN 2011000926W WO 2012162856 A1 WO2012162856 A1 WO 2012162856A1
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atom
leu
ser
gln
arg
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PCT/CN2011/000926
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Lishan Kang
Anders Svensson
Rune HARTMANN
Ole J. HAMMING
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Novo Nordisk A/S
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—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
    • C07K14/715—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2299/00—Coordinates from 3D structures of peptides, e.g. proteins or enzymes

Definitions

  • the present invention relates to models for cytokine receptor structures.
  • the invention relates to a model for the structure of the IL-21 receptor.
  • the immune response is based on a series of complex cellular interactions, which involves signaling between cells.
  • Soluble proteins known as lymphokines, cytokines, or monokines, play critical roles in controlling these cellular interactions.
  • Cytokines interact with cellular cytokine receptors, which are transmembrane structures which mediate signaling on binding of the cognate cytokine. Agonists and antagonists of cytokine receptors are useful in the modulation of cytokine activity, which can have a wide range of effects in immune therapy, cancer therapy and other medical applications.
  • IL-21 is a type I cytokine, which exerts pleiotropic effects on both innate and adaptive immune responses. It is mainly produced by activated CD4+ T cells, follicular T cells and Natural killer cells (NKT). In addition, recent evidence suggest that Th17 cells can produce high amount of IL-21.
  • IL-21 increases the cytotoxicity of CD8+ T cells and can promote proliferation of CD8+ cells in the presence of antigens.
  • 11-21 is induced by IL-6, a cytokine known to promote development of Th17 cells.
  • IL-21 acts on T helper cells in an autocrine manner promoting its own production and supporting differentiation of T-helper cells into Th17 cells.
  • IL-21 deficient mice show an impaired Th17 response.
  • IL-21 also acts on B-cells and increases antibody production; however, IL-21 is not essential for production of functional antibodies, whereas IL-21 R negative mice exhibit both reduced proliferation as well as impaired cytotoxicity of CD8+ cells.
  • a recent set of studies suggests that IL-21 produced by CD4+ cells is critical for the ability of CD8+ T cells to control viral infection.
  • IL-21 plays a complex role in autoimmune diseases.
  • the ability of IL- 21 to downregulate IgE production suggests that it could be used therapeutically against asthma and allergy; results from animal studies supports this view.
  • the ability of IL- 21 to promote Th17 development makes it a pro-inflammatory cytokine and a number of different IL-21 and IL-21 R inhibitors are currently investigated for use against a range of different autoimmune diseases.
  • IL-21 is a class I cytokine with a four helix bundle structure, arranged in an up-up-down-down topology typical for the class I cytokines.
  • IL-21 signals through a heterodimeric receptor complex consisting of the private chain IL-21 R and the common yC chain, the latter being shared by IL-2, IL-4, IL-7, IL-9, and IL-15.
  • the IL-21 R chain binds IL-21 with high affinity and provides the majority of the binding energy.
  • interaction with the yC chain is required for signaling and IL-21 mutants which bind IL-21 R but fail to interact properly with yC are potent antagonists of IL-21 signaling.
  • Both IL-2 and IL-15 employ a third receptor chain, IL-2Ra and IL- 15Ra respectively. These receptors are expendable for signaling, but works as high affinity component of the receptor complex capturing IL-2 or IL-15 and present it to IL-2RP after which recruitment of yC takes place.
  • Therapeutic intervention in cytokine pathways involves modulation of cytokine/receptor binding, by inhibiting or removing the cytokine, or by impeding binding to the receptor.
  • monoclonal antibodies specific for IL-21 are known in the art, such as from WO2007111714 and WO2010055366 (Zymo-Genetics, Inc.). Antibodies may be used to impede cytokine binding to its cognate receptor.
  • IL-2RP, IL-4Ra, and yC contain two type III fibronectin domains separated by a short linker.
  • the Fibronectin domains each contain 7 ⁇ - strands forming a sandwich like structure.
  • IL-2RP and IL-4Ra the two domains are bend at an angel of approximately 90° whereas the it is 120° in yC.
  • IL-2Rp:yC and IL-4Ra:yC form Y shaped structures on the surface of the cell with the cytokine binding in the fork.
  • the membrane proximal fibronectin domain contains a WSXWS motif, also known as the class I cytokine receptor signature motif. This motif is found in all Class I cytokine receptors. Previous attempts to clarify the functional role of this motif has failed, thus their function remains enigmatic.
  • a structural model of a class I cytokine receptor comprising a WSXWS motif, in which, in the tertiary structure, an R residue is located between the two W residues of the WSXWS motif to form a W-R-W zipper which is stabilized by a sugar bridge.
  • Models of class I cytokine receptors may be used to design or screen for drugs capable of modulating the interaction between cytokines and their receptors, or otherwise influencing receptor signaling.
  • Methods for screening and designing drugs based on a crystal structure derived model of a target are known in the art and described in more detail below.
  • the invention extends to both operator-controlled modeling and automated modeling techniques based on a cytokine receptor crystal structure-derived target.
  • Modeling may be carried out, in particular, on the regions of the target complex which are known to be required for cytokine-receptor interaction or receptor signaling.
  • the WSXWS motif forms a W-R-W zipper which is important in receptor signaling.
  • the model according to the invention advantageously comprises the WSXWS motif.
  • the present invention thus provides a crystal of a class I cytokine receptor and its cognate cytokine.
  • the crystal comprises the cytokine and the extracellular domain (ECD) of the cytokine receptor.
  • the cytokine and receptor are IL-21 and IL-21 R.
  • the crystal comprises IL-21 and the extracellular domain (ECD) of IL-21 R.
  • the present invention provides a crystal structure for a complex comprising a cytokine and its cognate receptor, such as IL-21 and IL-21 R.
  • Crystals according to the invention may be prepared using full-length cytokine and receptor polypeptides; preferably, however, the ECD of the receptor is employed in isolation.
  • the IL-21 R ECD is underglycosylated.
  • the IL-21 R polypeptide is underglycosylated by removing one or more glycosylation sites and/or one or more partially glycosylated sites.
  • the underglycosylated IL-21 R protein is deglycosylated by substituting amino acids at potential sites for N-linked glycosylation with Gin. If substitution with Gin leads to difficulties with protein expression, an Asp substituents may be used.
  • the polypeptide comprises the mutations N78Q, N85Q, N106D and N116Q.
  • IL-21 R is preferably not mutated at this position.
  • the crystal comprises atoms arranged in a spatial relationship represented by at least a portion of the structure co-ordinates of Table C.
  • atomic coordinates may be varied, without affecting significantly the accuracy of models derived therefrom; thus, although the invention provides a very precise definition of a preferred atomic structure, it will be understood that minor variations are envisaged and the claims are intended to encompass such variations.
  • the crystals provided in accordance with the present invention may be used to develop models useful for drug design and in silico screening of candidate molecules.
  • Models and/or atomic coordinates are advantageously stored on computer-readable media, such as magnetic or optical media and random-access or read-only memory, including tapes, diskettes, hard disks, CD-ROMs and DVDs, flash memory cards or chips, severs and the internet.
  • the computer capable of reading the medium according to the invention may be any suitable computer, such as a Windows®, Macintosh®, LINUX® or UNIX® - based system.
  • the invention accordingly provides a computer-readable medium having stored thereon a model of a class I cytokine receptor.
  • the present invention relates to a method of preparing the crystal of a class I cytokine receptor, according to the first aspect of the invention, comprising the steps of: (a) culturing host cells comprising an underglycosylated class I cytokine receptor protein; (b) purifying the underglycosylated cytokine recpetor protein; and (c) crystallising the underglycosylated cytokine receptor protein.
  • the cytokine receptor protein is underglycosylated as described above.
  • the cytokine receptor is an extracellular domain of a cytokine receptor; advantageously, the cytokine receptor is crystallised bound to its cognate cytokine.
  • the cytokine and receptor are IL-21 and IL-21R.
  • the present invention relates to a method of screening for a modulator of cytokine activity, comprising the use of a crystal according to the first aspect of the present invention.
  • the invention moreover provides modulators obtained from such a screen.
  • Fig 1 The N-linked glycosylation of Asn 54 is required for synthesis of the IL-21 R and bridges the two receptor domains.
  • Global view of the IL-21:IL-21R structure IL-21R is shown in lighter gray and IL-21 is shown in darker gray. Disulphide bridges are indicated by labels "CC”. Sugars are shown in gray sticks whereas the Trp 195 and Asn 54 are shown as sticks and labeled "T" and "N", respectively.
  • Fig 2. The carbohydrate chain attached to Asn 54 anchors at the class I cytokine receptor signature motif "WSXWS".
  • A 2F 0 -F C electron density map (at 1.4 sigma) around the sugar chain in IL-21 R. The amino acids Asn-54 and Trp-195 are labeled. 1.
  • B Close up of the anchor site, multiple hydrogen bonds are formed between the mannosylated Trp 195 and Arg 182 and the carbohydrate chain originating at Asn 54 . As seen Arg 182 , labeled ARG-182, is sandwich between the two Trp, labeled TRP-195 and TRP-198, of the WSXWS motif (Ser not shown), forming the W-R-W zipper descried in the text.
  • IL-21 binding pocket for methionine 70 of IL-21 R Residues of the IL-21 have been labeled with amino-acid type in upper case letters while IL-21 R amino-acid types with lower case letters. Electron density of the binding at the interface between IL-21 (indicated with darker gray carbon atoms) and IL-21 R (indicated with ligther gray carbon atoms). The binding pocket in IL-21 for Met-70 of IL-21 R.
  • Fig 4. Model of the IL-21 R/IL-21/yC complex.
  • A A predicted structure of the IL-21 R/IL-21/yC complex (see Table D). Site 1 represent the binding interface between IL-21 (medium gray) and IL-21 R (dark gray). Site 2 is the binding site between IL-21 (medium gray) and yC (light gray). Site 3 is the interaction between IL-21 R (light gray) and yC (medium gray). Note the surface complementarities.
  • B Effect of mutants in the yC binding region of IL-21. The yC is indicated in a surface representation while IL-21 is indicated in cartoon representation with particular residues shown in stick representation.
  • IL-21 residues displayed in black (Met-10, Arg-11 , lle-14, Gln-116, Lys-117, lle-119, His-120 and Leu-123) resulted in a substantial drop in affinity when mutated, ligther indicated residues (Met-7, Asp- 8, Glu-100 and Glu-109) had little or no effect upon IL-21 binding to yC.
  • Fig 5. The IL-21 R/IL-21 complex. Showing the residues in IL-21 and IL-21 R involved in binding to each other.
  • A The complex between IL-21, in dark, and IL-21 R, in light. Also indicated in stick representation are the residues in the IL-21/IL-21R interface and the glycosylation of the IL-21 R protein.
  • B The residues of IL-21 involved in the complex formation with IL-21 R are indicated by stick representation on top of a cartoon drawing of the IL-21 protein.
  • C The residues of IL-21 R involved in the complex formation with IL-21 are indicated by stick representation on top of a cartoon drawing of the IL-21 R protein.
  • Fig 6. Superimposition of IL-21 R/IL-21 with the structures of IL-2RB/yC/IL-2 and IL- 4RA yC/IL-4.
  • the structures of IL-2RA/IL-2RB/yC/IL-2 (Protein Data Bank accession code: 2B51) and IL-4RA/yC/IL-4 (PDB code: 3BPL) were superimposed onto the structure of IL-21 R/IL-21 in using SSM superimpose in coot.
  • model refers to a structural model such as a three dimensional (3D) structural model (or representation thereof) comprising a class I cytokine receptor.
  • Test compounds can be modeled that bind spatially and preferentially to the class I cytokine receptor - such as to bind spatially and preferentially to IL-21R.
  • the crystal model comprising class I cytokine receptor is built from all or a portion of the structural co-ordinates derived from a crystal of the cytokine receptor, for example the coordinates presented in Table C.
  • a class I cytokine or cytokine receptor is a cytokine, or receptor, falling within the group defined as such; see Liongue and Ward, BMC Evolutionary Biology 2007, 7:120. Most of the cytokine- binding receptors that function in the immune and hematopoietic systems belong to this receptor family. In addition, this family includes receptors for growth hormone and prolactin. There are conserved amino acid sequence motifs in the extracellular domain - 4 positionally conserved cysteine residues (CCCC) and a conserved sequence of Trp-Ser-X-Trp-Ser (WSXWS) where X is a nonconserved amino acid.
  • CCCC positionally conserved cysteine residues
  • WSXWS conserved sequence of Trp-Ser-X-Trp-Ser
  • the receptors consist of 2 polypeptide chains: a cytokine- specific subunit and a signal-transducing subunit which is usually not specific for the cytokine. In a few cases these receptors are trimers.
  • the signal transducing subunit is required for high affinity binding of the cytokine.
  • the IL-21 receptor, and IL-21 are for example as described in Parrish-Novak et al., Nature 408 (6808), 57-63 (2000), or Ozaki et al., Proc. Natl. Acad. Sci. U.S.A. 97 (21), 11439-11444 (2000).
  • the sequence and structure of this receptor, and its extracellular domain, are well known (see the examples for further information).
  • the cytokine receptor may be deglycosylated or underglycosylated.
  • underglycosylated means that one or more of the oligosaccharide chains covalently linked to amino acids in the glycosylated protein are no longer present.
  • deglycosylated implies that all of the oligosaccharide chains have been removed.
  • a crystal structure is defined as the particular repeating arrangement of atoms, molecules or ions throughout a crystal.
  • the position of these atoms can be defined with respect to a unit cell. As the entire crystal consists of repeating unit cells, this definition is sufficient to represent the entire crystal.
  • the atomic arrangement is expressed using coordinates.
  • atomic coordinates are usually expressed in terms of fractional coordinates, (x, y, z).
  • This coordinate system is coincident with the cell axes (a, b, c) and relates to the position of the atom in terms of the fraction along each axis.
  • a axis is 3.52 A long, the atom is (1.5/3.52) or 0.43 of the axis away from the origin.
  • it is (2.1/3.52) or 0.60 of the b axis and (2.4/3.5) or 0.68 of the c axis.
  • the fractional coordinates of this atom are, therefore, (0.43, 0.60, 0.68).
  • Co-crystallization involves the crystallization of two molecular entities.
  • a cocrystal is therefore a crystalline entity in which more than one molecular substance is incorporated into the unit cell. Crystals of salts, solvates etc. are not considered to be cocrystals.
  • IL-21 is a typical type I cytokine consisting of a four helical bundle with an up-up-down-down topology.
  • ECD-IL-21 R contains two fibronectin-lll domains each composed of 7 n-strands forming a sandwich like structure (fig. 1B). The two domains are connected by a short linker and bent at an angle of about 90°.
  • the N-terminal domain (D1) contains three disulfide bridges; one linking Cys 1 to Cys 90 , one linking Cys 6 to Cys 16 and one linking Cys 46 to Cys 62 .
  • the membrane proximal domain (D2) contains a "WSXWS” motif in the F'G' loop that is characteristic for class I cytokine receptors.
  • the two fibronectin-lll domains form a V like shape, with the binding site for the cytokine at the tip of the V.
  • the interaction between IL-21 R and IL-21 are described in detail below.
  • Trp195 and Trp198 of the WSXWS motif are situated at the surface of the molecule, in the final beta-strand before the trans-membrane helix.
  • the hydrophobic six membered part of their indol side chain is partly buried in the interior of the protein while the five member ring containing the nitrogen atom is exposed to the solvent.
  • Arg182 is held tightly in place in between the two Trp residues, with its head group exposed to the solvent.
  • IL-2Rp, IL-4Ra and yc all contain the "WSXWS" motif with an Arg sandwiched between the side chains of the two Trp.
  • the "WSXWS” motif in the primary structure thus turns out to be a W-R-W zipper in the tertiary structure as observed for the other class I cytokine receptors (15-17).
  • Trp195 was C-mannosylated at the C2 position of indole ring, the mannose is clearly visible in the density and has been modelled accordingly.
  • the carbohydrate chain attached to Asn54 in the DE loop is clearly visible in the density (fig 2A) and bridges the V formed by the two fibronectin domains of the ECD-IL-21R.
  • This bridge anchors at the W-R-W zipper by forming a hydrogen bonding network with Arg182 and the mannosylated Trp195 as well as Arg154 (Fig. 2B).
  • This sugar will in essence act as a stabilizing cross bar, transferring any strain put upon the D1 domain by the cytokine binding directly to the transmembrane part of the receptor.
  • This added rigidity could be essential for transmitting the signal across the cell membrane upon binding of IL-21.
  • it offers an explanation for the conservation of the "WSXWS" motif and the W-R-W zipper, found in class I cytokine receptors.
  • IL-2Rp, IL-4Ra and yc all have potential N-linked glycosylation sites on the D1 domain facing the D2 domain and they contain the conserved WSXWS motif.
  • the bridging sugar as well as the mannosylation of the first Trp in the WSXWS motif is conserved among class I cytokine receptors. Indeed, mannosylation of the WSXWS motif in the erythropoietin receptor (EPOR) has been demonstrated (19).
  • EPOR erythropoietin receptor
  • the role of the WSXWS motif is unclear in the prior art. It is not involved in ligand binding, neither does it seem to be involved in the hetero-dimerisation of the cytokine receptors.
  • X-linked severe combined immunodeficiency is a disorder where the function of the common yC chain is impaired by mutations.
  • X-SCID X-linked severe combined immunodeficiency
  • the interaction between IL-21 and ECD-IL-21 R is mediated by residues in the A and C helices as well as a small part of the CD-loop immediately preceding the C helix of IL-21.
  • the binding surface area is 2000 A 2 with extensive polar and apolar interactions. Seven different residues of IL-21 participate in polar interactions with nine residues of IL-21 R. There are 16 residues of IL- 21 forming van der Waals contact with 15 residues of IL-21 R. Arg9, Gin 12, Arg76 and Lys73 along with Ile16 form a pocket for et70 of IL-21 R, the main contributor to the binding surface in IL-21 R (Fig. 3).
  • the IL-21 binding residues of IL-21 R are located in the loops connecting the ⁇ -strands. Loops AB, CD, EF, B'C and F'G' as well as the linker all contain residues involved in binding. In IL-21 R Tyr36 in loop CD Met70 and Asp72 in the EF loop and Tyr129 in the B'C loop contribute the most to the binding surface. The most important loop is the EF loop, supplying 8 of the 19 amino acids of IL-21 R involved in binding IL-21.
  • the A and C helices are in nearly identical positions except for a small bend in the N terminal part of helix A in IL-21.
  • the D helices that bind yC superimpose well at the central part but are slightly tilted relative to one another.
  • Fig 4A We used the yC from the structure of the quaternary IL-2 complex, to generate a model of IL-21/IL-21R/yC (Fig 4A). There are no clashes between yC and the IL-21/IL-21 R structure and the model is in good agreement with the available biochemical data outlined below.
  • Fig. 4B shows a summary of previously published mutagenesis data on IL-21 binding to yC (24). Residues are color coded according to the observed effect of the mutation (see figure legend for details). According to our model Met7, Asp18 and Glu109 are lining the interaction surface but are still too far from yC for proper interaction, agreeing with the lack of effect when they are mutated. Changing Arg11 to Ala and Asp gives 3 and 12 fold reductions in the affinity of IL-21 towards yC respectively. Arg11 of IL-21 is at the interface between IL-21 R and yC and could thus interact with both. Mutating Ile14 and Ile119 to Ala has little effect on yC binding whereas a mutation to Asp greatly reduces binding.
  • Ile14 and Ile119 are close and interact with one another. They can furthermore bind a hydrophobic pocket made by Pro207 and Leu208 in yC explaining the detrimental effect of introducing a charged residue.
  • Gln116 to either Ala or Asp totally abolishes binding to yC.
  • several other side chains are tolerated at this position, including Phe, Lys, Asn and Glu.
  • the prominent role of Gln116 in IL-21 binding to yC and signaling is thus not a simple matter of size or functional group but the right combination of the two. His120 is found in sandwich between His159 (of yC) and the disulfide bridge between Cys160 and Cys209 (yC). This agrees well with mutational data, where introduction of a negative charge by mutating His120 to Asp results in loss of binding, whereas, changing His120 to Ala has a modest effect on yC affinity.
  • Leu123 One inconsistency between our model and the mutations is found at Leu123. Changing Leu123 is to alanine or aspartic acid results in a large drop in the affinity of IL-21 towards yC. As Leu 123 is outside the yC interface of IL-21 in our model we cannot easily explain this effect.
  • the term "crystal” can include any one of: a solid physical crystal form such as an experimentally prepared crystal, a 3D model based on the crystal structure, a representation thereof - such as a schematic representation thereof, a diagrammatic representation thereof, or a data set thereof for a computer.
  • the crystals of the present invention may be prepared by purifying IL-21 R and then co- crystallising the purified protein with purified IL-21.
  • the IL-21 R and IL-21 proteins may also be prepared by expressing a nucleotide sequence encoding the said proteins in a suitable host cell.
  • the crystals of the present invention are prepared by purifying underglycosylated IL-21 R and IL-21 proteins and then crystallizing the purified underdeglycosylated protein.
  • the underdeglycosylated IL-21 R and IL-21 proteins may also be prepared by expressing a nucleotide sequence encoding the underdeglycosylated IL-21 R and IL-21 proteins in a suitable host cell.
  • IL-21 R and IL-21 may be purified using various methods known to a person skilled in the art, for example, from conditioned media by affinity chromatography on a Sepharose-28-lisinopril affinity resin (Yu et al. 1997).
  • the protein may be quantified by amino acid analysis and assayed for activity using the substrate hippuryl-L-histidyl-L-leucine, as described in Ehlers, MRE, Chen, Y-_N, Riordan, JF (1991) Proc. Natl. Acad. Sci. USA 88, 1009-1013.
  • the purified IL-21 R and IL-21 proteins may be stored at 20 °C in 10 mM HEPES and 0.1 % PMSF. Concentration may be performed with the aid of a filtration system and the protein concentrate may be immediately used for crystallization purposes.
  • the protein concentrate may be crystallized using, for example, the vapor diffusion hanging or sitting drop method at a temperature of from about 1 °C to about 30 °C, preferably from about 4 °C to about 20 °C, more preferably at about 16 °C.
  • the crystallization temperature may be dependent on the additives present in the protein solution.
  • the best crystals for IL-21 R and IL-21 proteins are grown at 18 °C as sitting drops with a reservoir solution containing 500 ⁇ of 1 ,8-1 ,9 M di-Ammonium sulphate and 0.1 M Sodium IL- 21 Rtate at ph 5.5. Tantium bromide derivatives were obtained by adding 0.1 ⁇ of a 2 mM Ta 6 Br 2 solution. This was left for 2 hours at which point the crystals had turned green indicating uptake of Ta 6 Br 2 .
  • the crystal structure of the invention may contain a portion - such as at least 25%, at least 50%, at least 75%, or preferably at least 90%, at least 95%, at least 98%, or at least 99% - of the coordinates listed in Table C.
  • the crystal structure of the invention contains all of the co-ordinates listed in Table C.
  • the crystal is usable in X-ray crystallography techniques.
  • the crystals used can withstand exposure to X-ray beams used to produce diffraction pattern data necessary to solve the X-ray crystallographic structure.
  • glycoproteins that contain oligosaccharide chains covalently linked to certain amino acids. Glycosylation is known to affect protein folding, localization and trafficking, protein solubility, antigenicity, biological activity and half-life, as well as cell-cell interactions.
  • Protein glycosylation can be divided into four main categories mainly depending on the linkage between the amino acid and the sugar. These are N-linked glycosylation, O-linked glycosylation, C-mannosylation and GPI anchor attachments. N-glycosylation is characterised by the addition of a sugar to the amino group of an asparagine. In O-glycosylation, a sugar is attached to the hydroxyl group of a serine or threonine residue.
  • sequence motif Asn-_Xaa-_Ser/Thr (wherein Xaa is any amino acid other than Pro) has been defined as a prerequisite for glycosylation. Although rare, the sequence motif Asn-_Xaa-_Cys can also be an acceptor site.
  • N-glycans can be subdivided into three distinct groups called 'high mannose type', 'hybrid type', and 'complex type', with the common pentasaccharide core - Manp(alpha1 ,6)-(Manp(alpha1,3))-Manp(beta1l,4)-GlcpNAc (beta 1 ,4) GlcpNAc(beta 1 ,N)-Asn - occurring in all three groups.
  • glycosylation at Pro containing sites preventing glycosylation at Pro containing sites.
  • the negative influence of aspartic acid towards glycosylation can be ascribed to the negative charge on the side chain of this residue.
  • Ser/Thr is replaced by cysteine. While Ser replacement by Cys generally leads to decreased glycosylation, it has been shown (Kasturi 1995 J. Biol. Chem. 270: 14756-61) that substitution by Thr at a given potential glycosylation site can lead to increased glycosylation. This is in accordance with the model of hydrogen bonding being an important factor during the attachment of the precursor molecule to the protein.
  • glycosylation sites in a protein it has been estimated that glycosylation occurs only at one third of them, mostly at those sites where the surrounding amino acids allow the formation of a beta turn.
  • the ECD of the IL-21R contains 5 potential N-linked glycosylation sites, and purified recombinant ECD-IL-21R contains approximately 10 kDa glycans, to minimise glycosylation all potential N-linked glycosylation sites were individually mutated to Gin. After expression in HEK293 cells, all but one of the single mutated proteins showed a slight decrease in molecular weight. However, the Asn 54 to Gin mutation lead to an almost complete loss of protein secretion (fig. 1A). Residue Asn 54 was further substituted by Ala, Asp, and Gly. However, none of these mutants were secreted by HEK239 cells (data not show).
  • the glycan-minimized ECD-IL-21R (N780, N85Q, N106D, N116Q) was then constructed and expressed in HEK293 cells. This construct was used for crystallization.
  • IL-21R protein To deglycosylate the IL-21R protein, various methods known to a person skilled in the art may be used. Both chemical and enzymatic methods may be used for removing oligosaccharides from glycoproteins. Hydrazinolysis of glycoproteins (Kuraya, N & Hase (1992) J Biochem (Tokyo) 112:122-126), is capable of removing both N- and O-linked sugars, although this results in the complete destruction of the protein component and is therefore not suitable if recovery of the protein is desirable. Milder chemical methods such as trifluoromethanesulphonic acid (TFMS) may be used, however this may result in incomplete sugar removal and partial protein destruction. Other methods, including as site directed mutagenesis of glycosylated amino acidsm may also be used.
  • TFMS trifluoromethanesulphonic acid
  • enzymatic methods may be used which provide for complete sugar removal with no protein degradation.
  • PNGase F is an effective method of removing virtually all N-_linked oligosaccharides from glycoproteins (Tarentino & Plummer (1994). Methods in Enzymol 230:44- 57). The oligosaccharide is left intact and therefore suitable for further analysis (the asparagine residue from which the sugar is removed is deaminated to aspartic acid, the only modification to the protein).
  • Endoglycosidases include Endoglycosidase H (Kobata (1979) Anal Biochem 100:_1-14) and Endoglycosidase F (Trimble & Tarentino (1991) J. Biochem. 266:_1646-1651).
  • kits may also be used - such as the E-DEGLY kit (Sigma Aldrich, UK) and the GlycoFree Deglycosylation Kit (Glyco, Novato, USA) which removes both N- and O- linked glycans from glycoproteins.
  • IL-21 and IL-21 R being involved in a number of immune-related conditions, are targets for therapeutic intervention.
  • the present invention permits the use of molecular design techniques to design, select and synthesize chemical entities and compounds, including IL-21 modulating compounds, capable of binding to IL-21 R and/or IL-21, in whole or in part.
  • the present invention relates to a method of screening for a modulator of IL-21 activity wherein the method comprises the use of a cocrystal of IL21 :IL-21R.
  • the method comprises the steps of: (a) providing at least a portion of the structural co-ordinates of Table C; (b) employing at least a portion of the structural co-ordinates of Table C to design or select or synthesize a putative modulator of IL-21 activity; (c) assaying IL-21 activity in the presence of the putative modulator of IL-21 activity and IL-21 R.
  • the structural co-ordinates may be used to design compounds that bind to IL-21 R and modulate the interaction of the receptor with IL-21 itself, or influence signaling by the IL-21 receptor.
  • At least a portion of the structural co-ordinates of Table C and/or the putative modulator of IL-21 activity are provided on a machine-readable data storage medium comprising a data storage material encoded with machine readable data.
  • An IL-21 :IL-21R cocrystal may be probed with a variety of different chemical entities or test compounds to determine optimal sites for interaction between modulators of IL-21 activity and the receptor. For example, X-ray diffraction data collected from crystals grown in the presence of chemical entities or test compounds may allow the elucidation of how the chemical entities or test compounds interact with IL-21R. Molecules that bind to those sites can then be designed and synthesized and tested for their effect on IL-21 activity.
  • Small molecule databases or test compounds may be screened for chemical entities or compounds that can bind in whole, or in part, to IL-21 R.
  • the putative modulator of IL-21 activity is from a library of compounds or a database.
  • the quality of fit of such entities or compounds to the binding site may be judged by various methods - such as shape complementarity or estimated interaction energy ( eng, E. C. et al., J. Comp. Chem., 13, pp. 505-524 (1992)).
  • cytokine receptor proteins may crystallize in more than one crystal form
  • the structural co-ordinates of IL-21 R, or portions thereof may be particularly useful to solve the structure of other crystal forms of class I cytokine receptors. They may also be used to solve the structure of variants and complexes of such receptors.
  • the structural co-ordinates of IL-21 R are used to solve the structure of the crystalline form of any other class I cytokine receptor with significant structural homology to any functional domain of IL-21 R.
  • molecular replacement may be used.
  • the unknown crystal structure, whether it is another crystal form of IL-21 R, or another class I cytokine receptor may be determined using the IL-21 R structural co-ordinates of the present invention. This method will provide a more accurate structural form for the unknown crystal more quickly and efficiently than attempting to determine such information ab initio.
  • the IL-21 R crystal further comprises an entity bound to the IL-21 R protein or a portion thereof.
  • IL-21 R may be crystallised in complex with an entity that binds to it, such as IL-21 itself or mimic or an inhibitor of IL-21, which mimics IL-21 binding to IL-21 R.
  • the crystal structures of a series of such complexes may then be solved by molecular replacement or in combination with MAD (Multiwavelength Anomalous Dispersion) and/or MIRAS (Multiple Isomorphous Replacement with Anomalous Scattering) procedures - and compared with that of wild-type IL-21 R. Potential sites for modification within the binding sites of the enzyme may thus be identified. This information provides an additional tool for determining the most efficient binding interactions, for example, increased hydrophobic interactions, between IL-21 R and a IL-21 or a mimic or inhibitor thereof.
  • MAD Multiple Wavelength Anomalous Dispersion
  • MIRAS Multiple Isomorphous Replacement with Anomalous Scattering
  • the structures and complexes of IL-21 R may be refined using computer software - such as X- PLOR (Meth. Enzymol., vol. 114 & 115, H. W. Wyckoff et al., eds., Academic Press (1985)), MLPHARE (Collaborative computational project Number 4. The CCP4 Suite: Programs for Protein Crystallography (1994) Acta Crystallogr. D 50, 760-763) and SHARP [De La Fortelle, E. & Bricogne, G. Maximum-_likelihood heavy-_atom parameters refinement in the MIR and MAD methods (1997) Methods Enzymol. 276, 472-494).
  • the complexes are refined using the program CNS (Briinger et al.
  • the overall figure of merit may be improved by iterative solvent flattening, phase combination and phase extension with the program SOLOMON [Abrahams, J. P. & Leslie, A. G. W. Methods used in structure determination of bovine mitochondrial F1 ATPase. (1996) Acta Crystallogr. D 52,110-119].
  • the structural co-ordinates of the IL-21:IL-21R complex provided in this invention also facilitate the identification of related proteins or enzymes analogous to IL-21 or IL-21 R in function, structure or both, thereby further leading to novel therapeutic modes for treating or preventing immune-related diseases.
  • the design of compounds that bind to or modulate IL-21 R according to the present invention generally involves consideration of two factors.
  • Such conformational requirements include the overall three-dimensional structure and orientation of the chemical entity or compound in relation to all or a portion of a binding site of IL-21 R, or the spacing between functional groups of a compound comprising several chemical entities that directly interact with IL-21 R.
  • the potential modulating or binding effect of a chemical compound on IL-21 R may be analyzed prior to its actual synthesis and testing by the use of computer modeling techniques. If the theoretical structure of the given compound suggests insufficient interaction and association with IL-21 R, then synthesis and testing of the compound may be obviated.
  • the molecule may be synthesized and tested for its ability to bind to IL-21 R and modulate (eg. inhibit) using a suitable chemical or biological assay. In this manner, synthesis of inactive compounds may be avoided.
  • a modulating or other binding compound of IL-21 R may be computationally evaluated and designed by means of a series of steps in which chemical entities or test compounds are screened and selected for their ability to associate with IL-21 R.
  • a person skilled in the art may use one of several methods to screen chemical entities or test compounds for their ability to associate with IL-21 R and more particularly with the individual binding sites of IL-21 R. This process may begin by visual inspection of, for example, the active site on the computer screen based on the IL-21 R co-ordinates of the present invention. Selected chemical entities or test compounds may then be positioned in a variety of orientations, or docked, with IL-21 R. Docking may be accomplished using software such as Quanta and Sybyl, followed by energy minimization and molecular dynamics with standard molecular mechanics force fields - such as CHARMM and AMBER.
  • Specialized computer programs may also assist in the process of selecting chemical entities or test compounds.
  • suitable chemical entities or test compounds may be assembled into a single compound. Assembly may proceed by visual inspection of the relationship of the chemical entities or test compounds in relation to the structural co-ordinates of IL-21 R. This may be followed by manual model building using software - such as Quanta, Sybyl or O [Jones, T. A., Zou, J. Y., Cowan, S. W. & Kjeldgaard, M. Improved methods for building protein models in electron density maps and the location of errors in these models (1991) Acta Crystallogr. A 47, 110-119].
  • modulating or other IL-21 R binding compounds may be designed as a whole or de novo using either an empty binding site or optionally including some portion(s) of a known inhibitor(s).
  • Such compounds may be designed using programs that may include but are not limited to LEGEND (Nishibata and Itai (1991) Tetrahedron, 47, p. 8985) and LUDI (Bohm (1992) J. Comp. Aid. Molec. Design, 6, pp. 61-78).
  • the efficiency with which that compound may bind to IL-21 R may be computationally evaluated.
  • Specific computer software may be used to evaluate the efficiency of binding (eg. to evaluate compound deformation energy and electrostatic interaction) - such as QUANTA/CHARMM (Accelrys Inc., USA) and Insight ll/_Discover (Biosym Technologies Inc., San Diego, Calif., USA). These programs may be implemented, for instance, using a suitable workstation. Other hardware systems and software packages will be known to those persons skilled in the art.
  • substitutions may be made (eg. in atoms or side groups) to improve or modify the binding properties.
  • the substitutions may be conservative ie. the replacement group may have approximately the same size, shape, hydrophobicity and charge as the original group.
  • Such substituted chemical compounds may then be analyzed for efficiency of binding to IL-21 R by the same computer methods described above.
  • Test compounds and modulators of IL-21 etc. which are identified using the crystal and the methods of the present invention may be screened in assays. 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 Current screening technologies are described in Handbook of Drug Screening, edited by Ramakrishna Seethala, Prabhavathi B. Fernandes. New York, NY, Marcel Dekker, (2001).
  • NK92 a human NK cell line dependent on IL-2 or IL-21.
  • the NK92 cells will, when exposed to IL-21 , survive and proliferate, while cells cease proliferation and die within a few days without the stimulation of IL-21.
  • the proliferation rate of NK92 is closely correlated to the activity unit of IL-21. The higher activity of IL-21 that the cells are exposed to, the greater the rate of cellular proliferation. Proliferation of NK92 cells can therefore be used as an assay for biological activity of IL-21 and IL-21 variants.
  • the NK92 cells can be obtained from the American Type Tissue Collection and are cultured in MyeloCultTM (MyeloCultTM 5100, StemCell Inc, cat. nr. 05150) supplemented with 150 units/ml of IL-2 (Chemicon Cat.no. IL002), and penicillinstreptomycin; grown at 37 °C and 5% C02; and passaged every 48 h.
  • IL-2 starvation NK92 cells were plated in the absence of IL-2 for 12-16 h prior to hlL-2 or variant stimulation. Next 105 cells /80 ⁇ /well were seeded in 96-well plates, followed by adding 20 ⁇ of hlL-21 variant at different concentration.
  • the protein solution used for crystallization is at least 97.5% pure. More preferably, the protein solution used for crystallization is at least 99.0% pure. Most preferably, the protein solution used for crystallization is at least 99.5% pure.
  • 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 structural or functional mimetic, a peptide, a peptidomimetics, a derivatized test compound, a peptide cleaved from a whole protein, or a peptide synthesized 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, derivatives or combinations thereof.
  • the test compound may even
  • test compound will be prepared by recombinant DNA techniques and/or chemical synthesis techniques. Once a test compound capable of interacting with IL-21 R has been identified, further steps may be carried out to select and/or to modify the test compounds and/or to modify existing compounds, such that they are able to modulate IL-21 activity.
  • moduleating refers to preventing, suppressing, inhibiting, alleviating, restoring, elevating, increasing or otherwise affecting IL-21 activity, typically IL-21 activity exerted on or through IL-21 R.
  • IL-21 modulator may refer to a single entity or a combination of entities.
  • the IL-21 modulator may be an antagonist or an agonist of IL-21 , acting on IL-21 R.
  • IL-21 agonist means any entity, which is capable of interacting (eg. binding) with IL-21 R and which is capable of increasing a proportion of the IL-21 R that is in an active form, resulting in an increased biological response.
  • IL-21 antagonist means any entity, which is capable of interacting (eg. binding) with IL-21 R and which is capable of decreasing (eg. inhibiting) a proportion of the IL- 21 R that is in an active form, resulting in a decreased biological response.
  • the IL-21 modulators are antagonists of IL-21.
  • the modulator of IL-21 may be an organic compound or other chemical.
  • the modulator of IL-21 may be a compound, which is obtainable from or produced by any suitable source, whether natural or artificial.
  • the modulator of IL-21 may be an amino acid molecule, a polypeptide, or a chemical derivative thereof, or a combination thereof.
  • the modulator of IL-21 may be a polynucleotide molecule - which may be a sense or an anti-sense molecule.
  • the modulator of IL-21 may be an antibody.
  • the modulator of IL-21 may be designed or obtained from a library of compounds, which may comprise peptides, as well as other compounds, such as small organic molecules.
  • the modulator of IL-21 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 agent, a semi-synthetic agent, a structural or functional mimetic, a peptide, a peptidomimetic, a derivatized agent, 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 agent, an antibody, a natural or a non-natural agent, a fusion protein or equivalent thereof and mutants, derivatives or combinations thereof).
  • the modulator of IL-21 activity will be an organic compound.
  • the organic compounds will comprise two or more hydrocarbyl groups.
  • hydrocarbyl group means a group comprising at least C and H and may optionally comprise one or more other suitable substituents. Examples of such substituents may include halo-, alkoxy-, nitro-, an alkyl group, a cyclic group etc.
  • substituents may include halo-, alkoxy-, nitro-, an alkyl group, a cyclic group etc.
  • a combination of substituents may form a cyclic group. If the hydrocarbyl group comprises more than one C then those carbons need not necessarily be linked to each other. For example, at least two of the carbons may be linked via a suitable element or group.
  • the hydrocarbyl group may contain hetero atoms. Suitable hetero atoms will be apparent to those skilled in the art and include, for instance, sulphur, nitrogen and oxygen.
  • the modulator of IL-21 activity comprises at least one cyclic group.
  • the cyclic group may be a polycyclic group, such as a non-fused polycyclic group.
  • the modulator of IL-21 activity comprises at least the one of said cyclic groups linked to another hydrocarbyl group.
  • the modulator of IL-21 activity may contain halo groups, for example, fluoro, chloro, bromo or iodo groups.
  • the modulator of IL-21 activity may contain one or more of alkyl, alkoxy, alkenyl, alkylene and alkenylene groups - which may be unbranched- or branched-chain.
  • the modulator of IL-21 activity may be in the form of a pharmaceutically acceptable salt - such as an acid addition salt or a base salt - or a solvate thereof, including a hydrate thereof.
  • a pharmaceutically acceptable salt - such as an acid addition salt or a base salt - or a solvate thereof, including a hydrate thereof.
  • the modulator of IL-21 activity may be a structurally novel modulator of IL-21or IL-21 R.
  • the modulators of IL-21 activity may be analogues of known modulators of IL-21 - such as known inhibitors of IL-21 activity.
  • the IL-21 modulators have improved properties over those previously available, for example, fewer side effects.
  • the modulator of IL-21 activity may be a mimetic.
  • the modulator of IL-21 activity may also be chemically modified.
  • the modulator of IL-21 activity may be capable of displaying other therapeutic properties.
  • the modulator of IL-21 activity may be used in combination with one or more other pharmaceutically active agents.
  • combinations of active agents are administered, then they may be administered simultaneously, separately or sequentially.
  • mimetic relates to any chemical which includes, but is not limited to, a peptide, polypeptide, antibody or other organic chemical which has the same qualitative activity or effect as a known compound. That is, the mimetic is a functional equivalent of a known compound.
  • Modulators of IL-21 activity may exist as stereoisomers and/or geometric isomers - e.g. they may possess one or more asymmetric and/or geometric centers and so may exist in two or more stereoisomeric and/or geometric forms. The use of all the individual stereoisomers and geometric isomers, and mixture thereof is contemplated.
  • the modulator of IL-21 activity may be prepared by chemical synthesis techniques.
  • any stereocentres present could, under certain conditions, be racemized, for example if a base is used in a reaction with a substrate having an having an optical centre comprising a base-sensitive group. This is possible during e.g. a guanylation step. It should be possible to circumvent potential problems such as this by choice of reaction sequence, conditions, reagents, protection/deprotection regimes, etc. as is well- known in the art.
  • the compounds and salts may be separated and purified by conventional methods. Separation of diastereomers may be achieved by conventional techniques, e.g. by fractional crystallization, chromatography or H.P.L.C. of a stereoisomer ⁇ mixture of a compound of formula (I) or a suitable salt or derivative thereof.
  • An individual enantiomer of a compound of formula (I) may also be prepared from a corresponding optically pure intermediate or by resolution, such as by
  • a modulator of IL-21 activity that is a peptide can be synthesized by solid phase techniques, cleaved from the resin, and 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).
  • Synthesis of peptides may 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 431A Peptide Synthesizer (Perkin Elmer) in accordance with the instructions provided by the manufacturer. Additionally, the amino acid sequences comprising the modulator of IL-21 activity, 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 modulator of IL-21 activity.
  • the modulator of IL-21 activity may be a chemically modified modulator of IL-21 activity.
  • the chemical modification of a modulator of IL-21 activity may either enhance or reduce interactions between the modulator of IL-21 activity and the target - such as hydrogen bonding interactions, charge interactions, hydrophobic interactions, van der Waals interactions or dipole interactions.
  • the modulator of IL-21 activity may act as a model (for example, a template) for the development of other compounds.
  • IL-21 was expressed and purified from E. coli as previous described (1).
  • the ECD-IL-21 R was expressed in HEK293 cells and purified as described in (2).
  • the Complex of IL-21 and IL-21 R was formed by mixing IL-21 and IL-21 R at room temperature at a ratio of 1.5:1. Elevated levels of IL-21 were used, as IL-21 is most readily available. Furthermore, as IL-21 (15-16 kDa) is much smaller than IL-21 R (28kDa) it is more easily separated from the complex (43 kDA) after gel filtration.
  • the complex was loaded on to a HiLoad 16/60 Superdex 75 column (GFC) (GE Healthcare) and eluted with PBS (10 mM phosphate, 150 mm NaCI, pH 7.4). The fractions containing the complex were concentrated to 5 mg/ml using an Amicon Ultra-4 centrifugal filter device with a 10000 mw cut-off.
  • Crystals of selenomethionine IL-21 (semetll_21) in complex with IL-21 R were produced in the same way as wild type.
  • the phases from the Ta 6 Br 2 dataset were used to find sites in the semetlL21 dataset using anomalous difference fourier. This gave 50 initial hits. Manual inspection of these sites reviled that 20 were positioned as 3 sites with 8 fold symmetry with 4 sites missing.
  • the human IL-21 contains 4 methionines but from the available NMR structure(l) we knew that 2 of these (Met10 and Met118) are close in the structure. This makes it like that they would be seen as a single site at 6A.
  • the resulting map was of excellent quality given the resolution and the IL-21 R structure was built in de novo by repeated cycles of building and refinement in coot(6) and phenix respectively.
  • the final model contains 8 molecules of IL-21 RA and IL-21 forming the IL-21 R/IL-21 complex refined to a resolution of 2.9A.
  • the finished structure consists of residues 1-208 of IL21R and residues 2-81 and 89-123 of IL- 21 with small variations in the number of build residues between the 8 NCS related molecules.
  • the missing part of IL-21 is the loop connecting the C and D helices. This loop is not close to the IL-21 R binding site or the predicted yC binding site.
  • the 8 NCS related molecules adopt the same overall conformation but show small variations in some of the loops. These variations are however not located at the interface between IL-21 and IL-21 R or in the yC binding region of IL- 21 R. Furthermore the regions of IL-21 R interacting with the sugar chain are identical in all 8 molecules.
  • the sugar chain that has been build consists of 2 N-acetylglucoseamines, 1 fucose and 4 mannose residues. From the electron density the occupancies of the different sites are difficult to determine. Particularly mannose 575 might have occupancy less than 1 meaning that it might not be present in all the molecules in the crystal. The electron density is however sufficient for it to be build probably due to mannose 575 being held tightly in place when it is present. Mannose 575 might thus only be present in the sugar chain variant 2.
  • Table C The crystallographic coordinates of the IL-21/IL-21 R complex in PDB format (http://deposit.rcsb.org/adit/docs/pdb atom format.html).
  • Chain A show the coordinates of the IL-21 R molecule while the IL-21 chain is named B.
  • Atoms not in the list have not been able to be model due to missing density in the experimental electron density maps.
  • ATOM 14 N ASP A 3 29. .889 84. 735 28. .871 1. ,00 42. ,57 N
  • ATOM 63 N ASP A 9 32. .721 80. 855 48 .770 1. .00 48. .62 N
  • ATOM 114 CA ILE A 15 28. .270 83. ,455 43. .284 1. .00 28. , 95 C
  • ATOM 206 CD PRO A 25 14. ,314 86. 518 35. .279 1. 00 37. .90 C
  • ATOM 252 CA TRP A 32 29. ,903 69. 463 42. .019 1. 00 22. , 66 C
  • ATOM 283 CA GLN A 35 34. .835 61, .663 40 .550 1, .00 38. .78 C
  • ATOM 308 CD GLU A 37 29. , 654 56. ,359 41. .675 1. 00102. 18 C
  • ATOM 436 CA ASN A 54 22. 976 96. 047 36. .563 1. ,00 34. ,27 C
  • ATOM 442 O ALA A 55 21. ,942 95. ,800 31. .039 1. ,00 41. ,29 O
  • ATOM 444 CA ALA A 55 22. ,216 97. , 080 33. .037 1. , 00 47. .20 C
  • ATOM 456 CA HIS A 57 25. .460 91. .950 33. .840 1. .00 38. .07 C
  • ATOM 520 N ASP A 65 26. .584 73. ,931 51, .695 1. .00 17. .48 N
  • ATOM 570 CB MET A 70 38. , 621 68. .838 49. ,370 1. 00 29. 74 C
  • ATOM 588 N ASP A 73 38. .318 70, .336 44 , .557 1. .00 30. .77 N
  • ATOM 605 CA PHE A 75 31. .932 72, .471 40, .094 1. .00 30. .62 c
  • ATOM 636 O ILE A 79 21. .631 75. ,274 29, .848 1. ,00 37. , 88 O
  • ATOM 638 CA ILE A 79 22. .430 76. ,816 31. ,486 1. 00 29. 59 c
  • ATOM 684 CD GLN A 85 11. .550 70. ,259 25. .653 1. ,00 49. 68 C
  • ATOM 704 OG SER A 87 20. ,823 71. ,049 29. ,350 1. ,00 39. 80 0
  • ATOM 731 CA GLY A 91 32. .045 76. ,528 32. .700 1. ,00 31. 29 C
  • ATOM 760 CA LEU A 95 37. ,344 75. ,560 44. .191 1. ,00 24. 57 C
  • ATOM 787 N ILE A 99 38. .133 81. , 648 44. .738 1. .00 24. 70 N
  • ATOM 805 CA PRO A 101 36. .502 85. .668 50, .672 1. .00 25. , 14 C
  • ATOM 810 O ALA A 102 35. .581 88. , 137 53. .964 1. .00 29. 96 O

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Abstract

La présente invention concerne un co-cristal d'IL-21R et d'IL-21 basé sur son modèle structurel. L'invention concerne également un procédé de préparation d'un cristal de récepteur de cytokines de classe I.
PCT/CN2011/000926 2011-06-01 2011-06-01 Cristal de récepteur de cytokine WO2012162856A1 (fr)

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004024765A1 (fr) * 2002-09-12 2004-03-25 University Of Bath Structure de cristal d'une enzyme de conversion de l'angiotensine (ace) et ses utilisations

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004024765A1 (fr) * 2002-09-12 2004-03-25 University Of Bath Structure de cristal d'une enzyme de conversion de l'angiotensine (ace) et ses utilisations

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
OLSEN, S. K. ET AL.: "Crystal Structure of the Interleukin-15. Interleukin-15 Receptor alpha Complex Insights into Trans and Cis Presentation", JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 282, no. 51, 18 October 2007 (2007-10-18), pages 37191 - 37204 *

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