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WO1997008300A1 - Proteines cristallines de la famille zap - Google Patents

Proteines cristallines de la famille zap Download PDF

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Publication number
WO1997008300A1
WO1997008300A1 PCT/US1996/013918 US9613918W WO9708300A1 WO 1997008300 A1 WO1997008300 A1 WO 1997008300A1 US 9613918 W US9613918 W US 9613918W WO 9708300 A1 WO9708300 A1 WO 9708300A1
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WIPO (PCT)
Prior art keywords
atom
protein
zap
ligand
leu
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Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
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PCT/US1996/013918
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WO1997008300A9 (fr
Inventor
Marcos H. Hatada
Xiaode Lu
Ellen R. Laird
Jennifer L. Karas
Mark J. Zoller
Dennis A. Holt
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Ariad Pharmaceuticals Inc
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Ariad Pharmaceuticals Inc
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Priority to JP9510577A priority Critical patent/JPH11510061A/ja
Priority to AU69606/96A priority patent/AU6960696A/en
Priority to EP96930632A priority patent/EP0847443A1/fr
Publication of WO1997008300A1 publication Critical patent/WO1997008300A1/fr
Publication of WO1997008300A9 publication Critical patent/WO1997008300A9/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1205—Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • 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

Definitions

  • the invention relates to human ZAP-70, and in particular, to the region of ZAP-70 containing the tandem Src homoiogy-2 ("SH2") domains, to crystalline forms thereof, liganded or unliganded, which are particularly useful for the determination of the three-dimensional structure of the protein.
  • the three dimensional structure of the tandem SH2 region of ZAP provides information useful for the design of pharmaceutical compositions which inhibit the biological function of ZAP and other proteins of the ZAP famiiy, particularly those biological functions mediated by molecular interactions involving one or both SH2 domains.
  • Safe and effective immunosuppressive agents are required for the treatment of patients suffering from autoimmune disorders and for recipients of transplanted organs or tissues. For instance, in the absence of an effective immunosuppressive agent, patients often reject a transplanted organ, sometimes with fatal consequences.
  • the immunosuppressive agent must block the immune response, but must also be sufficiently well tolerated by the body to permit chronic application.
  • FK506 one compound with immunosuppressive activity
  • FK506 has been used to prevent rejection of transplanted livers.
  • severe kidney toxicity has been observed in patients receiving FK506, in some cases requiring kidney transplant following the liver transplant.
  • FK506 binds in a complex with two proteins, FKBP and calcineurin. FK506's immunosuppressive effects are due to the inhibition of calcineurin in T cells. However, since calcineurin is present and important in other cells, FK506 affects other ceils and tissues leading to undesired effects.
  • This invention concerns the region of human ZAP-70 spanning its two SH2 domains.
  • That region is the "ZAP tandem SH2 region” or simply "ZAP-NC”, since the region contains both the more M-terminal SH2 domain and the more C-terminal SH2 domain of human ZAP-70 (see FIG. 4).
  • the invention begins with obtaining crystals of human ZAP-NC, complexed or uncomplexed with various ligands, of sufficient quality to determine the three dimensional (tertiary) structure of the protein by X-ray diffraction methods.
  • renin has been modeled using the tertiary structure of endothiapepsin as a starting point for the derivation.
  • Model building of cercarial elastase and tophozoite cysteine protease were each built from known serine and cysteine proteases that have less than 35% sequence identity. The resultant models were used to design inhibitors in the low micromolar range. (Proc. Natl. Acad. Sci. 1993, 90, 3583).
  • Knowledge of the three-dimensional structure of a tandem SH2 region such as ZAP-NC provides a means for investigating the mechanism of action of the protein and tools for identifying inhibitors of its function.
  • SH2 domains are known to be involved in intramolecular and intermolecular interactions, usually protein-protein interactions, which are critical for biological activity of the SH2-bearing protein.
  • Knowledge of the three- dimensional structure of the tandem SH2 region allows one to design molecules capable of binding thereto, including molecules which are thereby capable of inhibiting the interaction of the tandem SH2 region with its natural iigand(s).
  • one object of this invention is to provide a composition
  • a composition comprising a protein in crystalline form having a peptide sequence derived or selected from that of a protein of the ZAP family.
  • the protein will comprise at least one, and preferably two SH2 domains, e.g., a protein containing the tandem SH2 region of ZAP-70, SYK or other related tandem SH2 containing protein, in the case of ZAP-70, the protein may comprise a peptide sequence spanning at least amino acid residues 3-279.
  • Such a crystalline composition may contain one or more heavy atoms, e.g., one or more lead, mercury, gold and/or selenium atoms, for instance.
  • Such a heavy atom derivative may be obtained, for example, by expressing a gene encoding the protein under conditions permitting the incorporation of one or more heavy atom labels (e.g. as in the incorporation of selenomethionine), reacting the protein with a reagent capable of linking a heavy atom to the protein (e.g. trimethyl lead acetate) or soaking a substance containing a heavy atom into the crystals.
  • a heavy atom derivative may be obtained, for example, by expressing a gene encoding the protein under conditions permitting the incorporation of one or more heavy atom labels (e.g. as in the incorporation of selenomethionine), reacting the protein with a reagent capable of linking a heavy atom to the protein (e.g. trimethyl lead acetate) or soaking a substance containing a heavy atom into the crystals.
  • a reagent capable of linking a heavy atom to the protein e.g. trimethyl lead acetate
  • the protein may be in the form of a complex with one or more ligand molecules, "ligand” being used in the broadest sense, referring to any substance capable of observable binding to the protein.
  • ligand being used in the broadest sense, referring to any substance capable of observable binding to the protein.
  • the peptide sequence of naturally occuring ligands (“ITAMs”, see below) for a number of SH2 domains is known and consensus sequence information on peptide ligands for SH2
  • peptide ligands of 15-19 residues derived in sequence from naturally occuring ligands for ZAP-70 or other SH2 domains may be used.
  • Those ligands typically contain one or two phosphorylated tyrosine residues.
  • one or both of such phosphorylated tyrosine moieties may be replaced by phosphotyrosine mimetic reagents, numerous examples of which are known in the art.
  • Illustrative crystalline compositions of this invention having various physicochemical characteristics are disclosed infra.
  • Preferred crystalline compositions of this invention are capable of diffracting x-rays to a resolution of better than about 3.5 A, and more preferably to a resolution of 2.6 A or better, and even more preferably to a resolution of 2.2 A or better, and are useful for determining the three-dimensional structure of the material. (The smaller the number of angstroms, the better the resolution.)
  • Crystalline compositions of this invention specifically include those in which the crystals comprise ZAP-family proteins characterized by the structural coordinates set forth in any of the accompanying Appendices or characterized by coordinates having a root mean square deviation therefrom, with respect to backbone atoms of amino acids listed in the Appendices, of 1.5 A or less.
  • Structural coordinates of a crystalline composition of this invention may be stored in a machine-readable form on a machine-readable storage medium, e.g. a computer hard drive, diskette, DAT tape, etc., for display as a three-dimensional shape or for other uses involving computer-assisted manipulation of, or computation based on, the structural coordinates or the three-dimensional structures they define.
  • a machine-readable storage medium e.g. a computer hard drive, diskette, DAT tape, etc.
  • data defining the three dimensional structure of a protein of the ZAP family, or portions or structurally similar homologues of such proteins may be stored in a machine-readable storage medium, and may be displayed as a graphical three-dimensional representation of the protein structure, typically using a computer capable of reading the data from said storage medium and programmed with instructions for creating the representation from such data.
  • This invention thus encompasses a machine, such as a computer, having a memory which contains data representing the structural coordinates of a crystalline composition of this invention, e.g. the coordinates set forth in our Appendices, together with additional optional data and instructions for manipulating such data.
  • data may be used for a variety of purposes, such as the elucidation of other related structures and drug discovery.
  • a first set of such machine readable data may be combined with a second set of machine-readable data using a machine programmed with instructions for using the first data set and the second data set to determine at least a portion of the coordinates corresponding to the second set of machine-readable data.
  • the first set of data may comprise a Fourier transform of at least a portion of the coordinates for ZAP or SYK proteins set forth in the Appendices hereto
  • the second data set may comprise X-ray diffraction data of a molecule or molecular complex.
  • one of the objects of this invention is to provide three-dimensional structural information on new complexes of ZAP family members with various ligands, as well as structural information on other tandem SH2 regions, previously unsolved individual SH2 domains, new ZAP family members and muteins or other variants of any of the foregoing.
  • molecular replacement uses a molecule having a known structure as a starting point to model the structure of an unknown crystalline sample. This technique is based on the principle that two molecules which have similar structures, orientations and positions in the unit cell diffract similarly. Molecular replacement involves positioning the known structure in the unit cell in the same location and orientation as the unknown structure.
  • the atoms of the known structure in the unit cell are used to calculate the structure factors that would result from a hypothetical diffraction experiment. This involves rotating the known structure in the six dimensions (three angular and three spatial dimensions) until alignment of the known structure with the experimental data is achieved. This approximate structure can be fine-tuned to yield a more accurate and often higher resolution structure using various refinement techniques. For instance, the resultant model for the structure defined by the experimental data may be subjected to rigid body refinement in which the model is subjected to limited additional rotation in the six dimensions yielding positioning shifts of under about 5%. The refined model may then be further refined using other known refinement methods.
  • molecular replacement may exploit a set of coordinates such as set forth in Appendix I, Appendix II or Appendix III to determine the structure of a crystalline co- complex of ZAP-NC, or a portion thereof, with a ligand other than the ⁇ 1 peptide.
  • a set of coordinates such as set forth in Appendix I, Appendix II or Appendix III to determine the structure of a crystalline co- complex of ZAP-NC, or a portion thereof, with a ligand other than the ⁇ 1 peptide.
  • a ligand other than the ⁇ 1 peptide
  • Another object of the invention is to provide a method for determining the three-dimensional structure of a protein containing at least one SH2 domain, or a co-complex of the protein with a
  • Homology modeling involves constructing a model of an unknown structure using structural coordinates of one or more related proteins, protein domains and/or subdomains. Homology modeling may be conducted by fitting common or homologous portions of the protein or peptide whose three dimensional structure is to be solved to the three dimensional structure of homologous structural elements. Homology modeling can include rebuilding part or all of a three dimensional structure with replacement of amino acids (or other components) by those of the related structure to be solved.
  • a set of coordinates defining the three dimensional structure of SYK-C complexed with the peptide Thr- pTyr-Glu-Thr-Leu which were obtained from evaluation of NMR data are set forth in Appendix IV.
  • Those coordinates may be stored, displayed, manipulated and otherwise used in like fashion as the ZAP-NC coordinates of Appendices I - III.
  • compositions of this invention provide a starting material for use in solving the three-dimensional structure of other members of the ZAP-70 family of proteins, notably SYK, as well as newly discovered proteins containing at least one SH2 domain and linking polypeptide (i.e., non-SH2 polypeptide) where the linking polypeptide has at least about 25% peptide sequence similarity, or preferably identity, to a portion (preferably at least six amino acids) of the ZAP-NC or SYK-NC inter-SH2 linking domain.
  • Sequence similarity may be determined using any conventional similarity matrix. See e.g. Dayhoff, M.O.; Schwartz, R.M.; Orcutt, B.C., Atlas of Protein Sequence and Structure 1979, 5, Suppl.
  • the structure defined by the machine readable data may be computationally evaluated for its ability to associate with various chemical entities.
  • chemical entity refers to chemical compounds, complexes of at least two chemical compounds, and fragments of such compounds or complexes.
  • a first set of machine-readable data defining the 3-D structure of a ZAP-family protein, or a portion or co-complex thereof is combined with a second set of machine-readable data defining the structure of a chemical entity or moiety of interest using a machine programmed with instructions for evaluating the ability of the chemical entity or moiety to associate with the ZAP-family protein or portion or complex thereof and/or the location and/or orientation of such association.
  • Such methods provide insight into the location, orientation and energetics of association of the ZAP family protein with such chemical entities.
  • Chemical entities that are capable of associating with the ZAP family member may inhibit its interaction with naturally occurring ligands for the protein and may inhibit biological functions mediated by such interaction. In the case of ZAP-70, such biological functions include activation of T cells during an immune response. Such chemical entities are potential drug candidates.
  • the protein structure encoded by the data may be displayed in a graphical format permitting visual inspection of the structure, as well as visual inspection of the structure's association with chemical entities.
  • more quantitative or computational methods may be used.
  • one method of this invention for evaluating the ability of a chemical entity to associate with any of the molecules or molecular complexes set forth herein comprises the steps of: a) employing computational means to perform a fitting operation between the chemical entity and a binding pocket or other surface feature of the molecule or molecular complex; and b) analyzing the results of said fitting operation to quantify the association between the chemical entity and the binding pocket.
  • This invention further provides for the use of the structural coordinates of a crystalline composition of this invention, or portions thereof, to identify reactive amino acids, such as cysteine residues, within the three-dimensional structure, preferably within or adjacent to a ligand binding site; to generate and visualize a molecular surface, such as a water-accessible surface or a surface comprising the space-filling van der Waals surface of all atoms; to calculate and visualize the size and shape of surface features of the protein or complex, e.g., ligand binding pockets; to locate potential H-bond donors and acceptors within the three- dimensional structure, preferably within or adjacent to a ligand binding site; to calculate regions of hydrophobicity and hydrophilicity within the three-dimensional structure, preferably within or adjacent to a ligand binding site; and to calculate and visualize regions on or adjacent to the protein surface of favorable interaction energies with respect to selected functional groups of interest (e.g.
  • design or select compounds capable of specific covalent attachment to reactive amino acids e.g., cysteine
  • complementary characteristics e.g., size, shape, charge, hydrophobicity/hydrophilicity, ability to participate in hydrogen bonding, etc.
  • the structural coordinates of the ZAP family protein, or portion or complex thereof are entered in machine readable form into a machine programmed with instructions for carrying out the desired operation and containing any necessary additional data, e.g. data defining structural and/or functional characteristics of a potential ligand or moiety thereof, defining molecular characteristics of the various amino acids, etc.
  • One method of this invention provides for selecting from a database of chemical structures a compound capable of binding to a ZAP family protein.
  • the method starts with structural coordinates of a crystalline composition of the invention, e.g., coordinates defining the three dimensional structure of a ZAP famiiy protein or a portion thereof. Points associated with that three dimensional structure are characterized with respect to the favorability of interactions with one or more functional groups.
  • a database of chemical structures is then searched for candidate compounds containing one or more functional groups disposed for favorable interaction with the protein based on the prior characterization. Compounds having structures which best fit the points of favorable interaction with the three dimensional structure are thus identified.
  • a first set of machine-readable data defining the 3D structure of a ZAP- family protein, or a portion or protein-iigand complex thereof is combined with a second set of machine readable data defining one or more moieties or functional groups of interest, using a machine programmed with instructions for identifying preferred locations for favorable interaction between the functional group(s) and atoms of the protein.
  • a third set of data i.e. data defining the location(s) of favorable interaction between protein and functional group(s) is so generated.
  • That third set of data is then combined with a fourth set of data defining the 3D structures of one or more chemical entities using a machine programmed with instructions for identifying chemical entities containing functional groups so disposed as to best fit the locations of their respective favorable interaction with the protein.
  • Compounds of the structures selected or designed by any of the foregoing means may be tested for their ability to bind to a ZAP famiiy protein, inhibit the binding of a ZAP famiiy protein to a natural or non-natural ligand therefor, and/or inhibit a biological function mediated by a ZAP family member.
  • This invention also provides peptidomimetic methods for designing a compound capable of binding to a ZAP family protein.
  • One such method involves graphically displaying a three- dimensional representation based on coordinates defining the three-dimensional structure of a ZAP family protein or a portion thereof complexed with a ligand. Interactions between portions of a ligand and the protein are characterized in order to identify candidate moieties for replacement.
  • One or more portions of the ligand which interact with the protein may be replaced with substitute moieties selected from a knowledge base of one or more candidate substitute moieties, and/or moieties may be added to the ligand to permit additional interactions with the protein.
  • the computational approaches and structural insights disclosed herein also permit the design or identification of molecules with reduced capability, or substantial / ⁇ ability, to bind to a ZAP family protein.
  • the goal of such efforts are inhibitors of those other SH2- mediated interactions which lack ZAP-family mediated activities, such as immunosuppression, which in that context, would be undesired side effects.
  • FIG. 1A Backbone ribbon representation of the overall fold of the complex of ZAP-NC and the ⁇ l peptide oriented such that the N-terminal SH2 domain is on the left-hand side of the figure, the C-terminal SH2 domain is on the right-hand side of the figure and the inter-SH2 domain region is in the middle, dropping toward the bottom of the figure. Secondary structural elements are labeled according to the convention for SH2 domains. 20 An ⁇ -carbon trace of the peptide is included. All elements are labeled in ZAP-N; in ZAP-C, only the central sheet and helices are labeled. Termini of the protein and peptide are denoted by N and C.
  • Loop regions are named for the secondary elements which they connect, i.e., the BC loop connects strands B and C.
  • the N terminus of the peptide is at the right-hand side of the figure.
  • the peptide consists of 19 residues, starting at residue 48 of the mature ⁇ subunit of the T cell receptor (TCR).
  • Phosphotyrosines are at relative positions 4 and 15. Definition of least squares planes fitted to the main chain atoms of each pYXXL motif generates a pair of planes at an angle of 120X Due to the staggered orientation of the SH2 domains, the pYXXL motifs are separated by an "S"-shaped segment of peptide. This sequence contains nearly one full turn of an ⁇ -helix between residues ⁇
  • FIG. 1 B The BC loop, from ⁇ B5 to ⁇ C3, of ZAP-N may be superimposed with the corresponding residues of the Lck SH2:middle T complex 20 and the relative position of the BC loops examined when the structures are fitted according to secondary structural elements. Superimposition of the backbone atoms of the loops results in an r.m.s. deviation of 0.65 A. Similar results are observed with the BC loop of ZAP-C. In complexes of phosphopeptides with isolated SH2 domains, the BC loop contributes nearly half of the direct hydrogen bonds to the phosphate group. For both SH2 domains of ZAP-NC, the BC loop is extended such that several waters are mediating contact between the loop and the phosphate group.
  • FIG. 2 Sequence alignments [SEQ ID NOS 21 - 25] for selected SH2 domains. 33 Boxed areas indicate segments used for measuring "full" backbone r.m.s. deviation; unboxed areas are excluded from the calculation due to the presence of gaps for one or more of the sequences. Notation below the alignments indicate structurally conserved regions, and use the previously reported nomenclature; 20 these regions were used for the "core" r.m.s. deviation calculation. Thin-lined boxed areas within ZAP-N and ZAP-C indicate the secondary structural elements in
  • FIG. 3 Phosphotyrosine binding sites.
  • the phosphotyrosine residues are oriented in a similar fashion in each figure to facilitate direct comparison, a, Selected residues for the ⁇ pTyr 4 association with ZAP-C.
  • Direct hydrogen bonds to the phosphate group of ⁇ pTyr 4 are indicated by dashed lines.
  • Crystallographic waters are indicated as spheres; waters labeled 551 , 555, and 556 make bridging contacts between the phosphate and the SH2 domain, water 622 forms a bridge to ⁇ -j .
  • b Selected residues in close association with ⁇ pTyr 15.
  • dashed lines indicate direct hydrogen bonds to the phosphate group.
  • Tyr 238 and Lys 242 from ZAP-C complete the hydrogen-bonding network of the phosphate.
  • Waters are represented as spheres; all of them are involved in salt-bridging the phosphotyrosine to the SH2 domains.
  • residues that form the pocket have been omitted for clarity, c.
  • the interface between the SH2 domains involves an extensive network of hydrogen bonds. Interactions involving ⁇ pTyr 15
  • Arg 41 has three hydrogen-bonding contacts to ZAP-C. This is the first of three residues in the BC loop of ZAP-N that form an artificial parallel sheet with strand F in ZAP-C. Only one of the three hydrogen-bonding contacts involves main chain atoms exclusively.
  • FIG. 4 Schematic view of ZAP-70 bound to the ⁇ -
  • Activation of T cells is initiated by association of the T cell receptor (TCR) with a peptide antigen bound to the major histocompatibility complex (MHC) on an antigen-presenting cell.
  • TCR-MHC association stimulates phosphorylation of T cell receptor subunits on tyrosi ⁇ es within the ITAMs (most likely by the Src family PTKs, Lck or Fyn).
  • ZAP-70 binds to the phosphorylated ITAM via its tandem SH2 domains (amino acids 1-259) in an orientation such that the N-SH2 domain binds to the C-proximal pYXXL motif and the C-SH2 domain binds the
  • N-proximal pYXXL motif N-proximal pYXXL motif.
  • Proposed positions of the other domains of ZAP-70 referred to as interdomain B (amino acids 260-310) and catalytic domain (amino acids 311 -620) are illustrated.
  • Two ZAP-70 molecules could bind to the activated TCR complex since the ⁇ subunit is present as a disulfide-linked dimer.
  • the primary determinants of binding are the phosphotyrosine and leucine residues of two pYXXL sequences within an ITAM.
  • the structure reveals a unique binding pocket for the pY of the C-proximal pYXXL motif in the interface between the two SH2 domains.
  • the crystal structure reveals that interdomain A forms a coiled-coil helical structure. This domain may participate in positioning the two SH2 domains for association with ITAMs, and in communicating structural changes to interdomain B and/or the kinase domain upon receptor engagement.
  • FIG. 5 depicts a binding curve of a doubly-phosphorylated ⁇ -1 :ZAP-NC complex, with associated Scatchard plot of the data, as determined by fluorescence polarization.
  • FIG. 6 depicts the ZAP-NC: ⁇ 1 complex encased in a gridded box for receptor site mapping. Only the backbone of ZAP-NM and the peptide are illustrated. Colors may be indicated as follows: red and blue for oxygen and nitrogen atoms, respectively; yellow for carbon atoms of the peptide; cyan, orange and green for N-terminal SH2 domain, the inter-SH2 spacer and the C-proximal SH2 domain carbon atoms, respectively. Note that the box encompasses space that is occupied by the peptide, as well as several interfacial regions of the SH2 domains.
  • FIG. 7 depicts a representative site contour map of ZAP-NC.
  • FIG. 7A A selected region of the receptor map for ZAP-NC plus an amino cation probe contoured at -10 kcal/mol. A pocket on the protein surface is indicated by a solvent-accessible surface.
  • FIG. 7B The individual amino acids that are in close proximity to the contour map are shown explicitly. The position of the contour indicates that strong interactions will be achieved between a hydrogen-bond donating moiety and the main chain carbonyl oxygens of Arg 192, Glu 194, and Thr 197, as well as the side chains of Gin 195 and Tyr 198.
  • FIG. 8 depicts a computer system.
  • FIG. 9 depicts storage media of this invention.
  • T cell recognition of antigen-presenting cells initiates a cascade of intracellular processes that ultimately result in changes in gene expression, the production of secreted mediators, and cellular proliferation.
  • TCR T cell receptor
  • the intracellular portion of each subunit includes one to three peptide sequences that contain the motif YXX( )X(7-8)YXX(L/I), where X is variable.
  • these immunoreceptor tyrosine activation motifs, or ITAMs become phosphorylated on tyrosine residues and in this modified form, provide binding sites for downstream signaling proteins.
  • the TCR has no intrinsic protein tyrosine kinase (PTK) activity, however members of both the Src family and the SYK/ZAP-70 family of PTKs are implicated in the functioning of antigen receptors. 4 Current evidence indicates that Src family kinases phosphorylate the ITAMs of the
  • ZAP-70 then associates with the doubly-phosphorylated ITAMs of the ⁇ and CD3 ⁇ chains through its SH2 domains 5 and is itself phosphorylated during early T cell activation.
  • ZAP-70 (£ associated protein) is a 70 KDa protein tyrosine kinase that is expressed exclusively in T cells and NK cells.
  • ZAP-70 is known to play a critical role in T ceil activation. Genetic alterations in the ZAP-70 gene that cause loss of expression of ZAP-70 in humans prevent antigen activation of CD4 + T cells, inhibit maturation of CD8 + T cells, and lead to severe combined immunodeficiencies.
  • 8 * 9 ZAP-70 binding to the TCR is believed to be essential for signal transduction since peptides that block the association of ZAP-70 with the ⁇ chain also
  • ZAP-70 is an ideal target for the development of novel immunosuppressive therapies.
  • the first 259 residues of ZAP-70 consist of two SH2 domains that are connected by a 65 residue segment and are followed by a second connecting region and a catalytic domain. 7 SH2 domains consist of approximately 100 amino acids. Their role in the specific recognition of tyrosine-phosphorylated proteins is integral to a variety of intracellular signaling events (recently reviewed in 1 1 - 12 ). Several SH2 domains have been demonstrated to retain the ability to bind with high affinity to short peptides that contain phosphotyrosine (pY) when expressed as isolated proteins. Selectivity for isolated SH2 domains is dependent upon recognition of residues immediately C-terminal to the phosphorylated tyrosine (pY+n).
  • ZAP-NC is one of a number of proteins that contain two src-homology 2 (SH2) domains.
  • SH2 domain a protein sequence that contains two src-homology 2 (SH2) domains.
  • the presence and boundaries of an SH2 domain in a protein sequence can be identified by using a computer alignment program that identifies amino acid sequence homology to a known SH2 domain.
  • SH2 domain amino acids between 140-255 of Src are used for such analyses, but SH2 domains from other proteins can be used as well.
  • the alignment method typically used by such programs is the Needleman-Wunch alignment. See e.g., "A General Method Applicable to the Search for Similarities in the Amino Acid Sequence of Two Proteins.” Needlman, S.B.; Wunch, C.D. J. Mol. Biol.
  • SH2 domains have been identified in a large and growing number of proteins, some of which contain multiple SH2 domains. For example, tandem SH2 regions are present in human ZAP-70 (spanning amino acids 1 -259), human SYK (spanning amino acids q - z), PLC ⁇ , PI3K, rasGAP, SH-PTP1 , and
  • ZAP-NC as a glutathione-S-transferase (GST) fusion protein.
  • GST glutathione-S-transferase
  • the cDNA encoding residues 1 -259 from human ZAP-70 1 ° was cloned into the pGEX2T expression vector 41 and transformed into E. coli BL21 or E. coli B834.
  • the resulting construct produced a fusion protein of GST-ZAP-NC linked by a polypeptide segment containing the sequence -LVPRGS- which comprises a thrombin cleavage site.
  • the selenomethionyl (SeMet) ZAP-NC was produced using the auxotrophic strain of E. coli 834 42 with the selenomethionine replacing methionine in a defined media.
  • the GST-ZAP-NC fusion protein was isolated using glutathione agarose and then cleaved with thrombin. Cleavage yields two polypeptides, the GST and ZAP-NC.
  • the ZAP-NC polypeptide contains two extra amino acids (Gly-Ser) at the amino terminus from the linker segment of the pGEX2T expression vector. These two extra amino acids were shown to have no functional effect on ZAP-NC binding to peptide ligands.
  • ZAP-NC was separated from GST by binding ZAP-NC to a phosphotyrosine agarose column and eluting with a salt gradient. Subsequently, ZAP-NC was further purified on a phenyl sepharose column.
  • ZAP-NC:peptide ligand complexes were formed by mixing two-fold excess of peptide and purified ZAP-NC, then subjecting the mixture to chromatography using a superdex 75 gel filtration column. Fractions containing the purified ZAP-NC:peptide complex were combined and used for subsequent crystallization experiments.
  • ZAP-NC proteins may also be used, including ZAP-NC proteins truncated at the N- terminus and/or C-terminus to contain just the SH2 homology boundaries.
  • the protein may be extended at the C-terminus to include additional amino acids extending to include additional domains (spacer B) up to the entire ZAP-70 protein (amino acids 1-620).
  • tandem SH2 regions especially from human and non-human ZAP famiiy members, including proteins such as human SYK, may be prepared and used in analogous fashion to that described herein.
  • the tandem SH2 protein may be produced in E. coli using T7, maltose-binding protein fusion (MBP), with epitope tags (His6, HA, myc, Flag) included or cleaved off.
  • MBP maltose-binding protein fusion
  • epitope tags His6, HA, myc, Flag
  • Baculoviral expression may be used, e.g. using pVL1393 or derivatives, for tandem SH2 protein, fused (or not) to epitope tag or fusion partner such as GST.
  • Conventional materials and methods for expression in mammalian, yeast or other ceils may also be used.
  • Peptide ligands for co-crystallization with ZAP-NC or other tandem SH2 proteins may be prepared using conventional methods, containing peptide sequences based on naturally occurring ITAM sequences such as ITAM sequences derived from the T cell receptor ⁇ 1 , ⁇ 2, ⁇ 3, ⁇ , ⁇ or ⁇ subunits or from the ⁇ or ⁇ subunits of the IgE receptor, for example.
  • ITAM sequences derived from the T cell receptor ⁇ 1 , ⁇ 2, ⁇ 3, ⁇ , ⁇ or ⁇ subunits or from the ⁇ or ⁇ subunits of the IgE receptor, for example.
  • ITAM sequences derived from the T cell receptor ⁇ 1 , ⁇ 2, ⁇ 3, ⁇ , ⁇ or ⁇ subunits or from the ⁇ or ⁇ subunits of the IgE receptor for example.
  • such ligands will contain the 15 amino acid minimal ITAM sequence YXXLXXXXXXXYXXL [SEQ ID NO 1
  • the C-terminus may be a free carboxylate or may be amidated or otherwise modified.
  • Tyrosines may (each) be phosphorylated (pTyr). Altematively, difluourophosphonoTyr, phosphonomethyl phenylalanine, hemi-phosphorylated Tyr, or other pTyr mimetics may be used in place of pTyr.
  • the ligand may contain amino acid replacements, insertions or deletions with respect to a naturally occurring ITAM sequence.
  • hybrid peptide-nonpeptide ligands and non ⁇ peptide ligands may also be used. Examples of such ligands are depicted in Table 1.
  • Crystallization Crystallization experiments were conducted using a sparse matrix screening approach, in the case of ZAP-NC crystals, beginning with a Crystal Screen 1 kit (Hampton Research, Riverside, CA). Crystals containing SYK-NC were obtained as described below. In the case of ZAP-NC, best results were obtained using protein stabilized in 0.5 M NaCI, followed by removal of salt by dialysis prior to the crystallization experiments. Special handling of that sort was not necessary for SYK-NC, but may be useful for other tandem SH2 containing proteins.
  • ITAM peptide (Ligand 5, Table 1 ) were grown from polyethylene glycol (PEG) 4000.
  • the structure was elucidated by multiple isomorphous replacement at 1.9 A resolution. It was not possible to solve the structure by molecular replacement alone using coordinates for previously determined SH2 domains. This was due to the low sequence identity with other SH2 domains and between the two ZAP domains, as well as to the presence of the 65 residue interdomain region.
  • the details of crystallization, data collection, multiple isomorphous replacement (MIR), and refinement are described below.
  • peptide was concentrated to 30 mg/ml in 20mM Tris, pH 8.5, 200mM sodium chloride and 20mM dithiothreitol.
  • the complex was treated with 4mM trimethyllead acetate (TML).
  • Crystals were obtained by vapor diffusion in hanging drops containing 13.5 mg/ml complex and 10% PEG 4000, 50mM sodium citrate, 100mM ammonium acetate, 0.005% sodium azide and 20mM dithiothreitol, pH 6.2, over reservoirs of 20% PEG 4000 and 20mM dithiothreitol.
  • the crystals are monoclinic (P2-
  • IgE ⁇ TAM 15mer [SEQ ID NO 3] Ac.pTyr.Thr.Gly.Leu.Ser.Thr.Arg.Asn.Gln.Glu.Thr.pTyr.Glu.Thr.Leu.NH2
  • Trp Tyr Phe Gly Lys Ile Thr Arg Arg Glu Ser Glu Arg Leu Leu Leu 1 5 10 15
  • MOLECULE TYPE protein
  • FRAGMENT TYPE internal
  • phosphotyrosine pTyr, pY
  • F2Pmp difiuorophosphonomethylphenylaianine
  • Fmoc Fluorenylmethyloxycarbonyl
  • N-methyl nm
  • acetyl Ac
  • Diffraction data were collected with an Rigaku R-AXIS II area detector with graphite monochromated Cu K ⁇ X-rays. Diffraction data were collected as 2° oscillation images and reduced to integrated intensities with DENZO. 43 Scaling parameters for each image were calculated with ROTAVATA 44 and applied with AGROVATA. 44 Data sets were collected for all SYK and ZAP crystals (with and without bound ligands) at -160°C except for those collected at room temperature for crystals containing ZAP-NC protein containing lead.
  • SeMet ZAP-NC was crystallized with TML under the same conditions and data collected. Positions of the lead and selenium atoms were determined from the difference Patterson function. Anomalous dispersion measurements were included for both datasets. Heavy atom parameters were refined, and phases were obtained at 2.8 A with the program MLPHARE. 44 The MIR phases were improved with the program DM 44 with a combination of solvent flattening/histogram mapping and phase extension to 2.0 A. Electron density maps with MLPHARE and DM phases were calculated, and the polypeptide chain model was built with the program O 45 . SIGMAA 44 was used to perform several cycles of phase combination using partial model and experimental phases. Least squares refinement with simulated annealing was done using X-PLOR.
  • the current model has all residues from Asp 3 to Asn 256 of the protein, all 19 peptide residues of zeta-1 , and 113 water molecules, plus one lead and three selenium atoms. TML is bound to Cys 1 17. See Table 2.
  • compositions of this invention may be obtained as described in detail herein.
  • Appendix I, Appendix II and Appendix III set forth the structural coordinates, in PDB format, for crystalline compositions comprising ZAP- NC: ⁇ 1 "monomer” (one molecule of complex per unit cell), ZAP-NC: ⁇ 2, and ZAP-NC: ⁇ 1 "dimer” (two molecules of complex per unit cell).
  • This invention encompasses crystalline compositions containing a ZAP family protein having a region characterized by structural coordinates set forth in Appendices I, II or lil, or by coordinates having a root mean square deviation therefrom of less than about 1.5 A, preferably
  • Rc ⁇ I lFph ⁇ Fp
  • the Molecular Similarity application permits comparisons between different structures, different conformations of the same structure, and different parts of the same structure.
  • the procedure used in Molecular Similarity to compare structures is divided into four steps: (1 ) load the structures to be compared; (2) define the atom equivalences in these structures; (3) perform a fitting operation; and (4) analyze the results.
  • Each structure is identified by a name.
  • One structure is identified as the target (i.e., the fixed structure); all remaining structures are working structures (i.e., moving structures). Since atom equivalency within QUANTA is defined by user input, for the purpose of this invention we define equivalent atoms as protein backbone atoms (N, C ⁇ , C and O) for all conserved residues between the two structures being compared and consider only rigid fitting operations.
  • the working structure is translated and rotated to obtain an optimum fit with the target structure.
  • the fitting operation uses a least squares fitting algorithm that computes the optimum translation and rotation to be applied to the moving structure, such that the root mean square difference of the fit over the specified pairs of equivalent atom is an absolute minimum. This number, given in angstroms, is reported by QUANTA.
  • root mean square deviation means the square root of the arithmetic mean of the squares of the deviations from the mean. It is a way to express the deviation or variation from a trend or object.
  • root mean square deviation defines the variation in the backbone of a protein from the backbone of a protein of this invention, such as
  • ZAP-NC as defined by the structural coordinates of Appendix I, Appendix II or Appendix lil and described herein.
  • least squares refers to a method based on the principle that the best estimate of a value is that in which the sum of the squares of the deviations of observed values is a minimum.
  • the structural coordinates generated for a crystalline substance of this invention e.g. the structural coordinates of ZAP-NC, ⁇ 1 , ⁇ 2, or the various complexes as depicted in Appendix I, Appendix II or Appendix III
  • Midas (University of California, San Francisco)
  • MidasPlus (University of California, San Francisco)
  • Chem-3D (Cambridge Scientific) Chain (Baylor College of Medicine) O (Uppsala University) GRASP (Columbia University) X-Plor (Molecular Simulations, Inc.; Yale University)
  • VMD Universality of lllinois/Beckman Institute
  • Sculpt Interactive Simulations, Inc.
  • a machine-readable storage medium comprising a data storage material encoded with machine readable data which, when using a machine programmed with instructions for using said data, e.g. a computer loaded with one or more programs of the sort identified above, is capable of displaying a graphical three-dimensional representation of any of the molecules or molecular complexes described herein.
  • Machine-readable storage media comprising a data storage material include conventional computer hard drives, floppy disks, DAT tape, CD-ROM, and other magnetic, magneto-optical, optical, floptical and other media which may be adapted for use with a computer.
  • a machine-readable data storage medium that is capable of displaying a graphical three-dimensional representation of a molecule or molecular complex that is defined by the structural coordinates of a protein of the ZAP famiiy, e.g. ZAP-NC or SYK-NC, or portion thereof, and in particular, structural coordinates of ZAP-NC: ⁇ 1 or ZAP-NC: ⁇ 2 set forth in Appendix I, Appendix II or Appendix III (or derivatives thereof such as zapNC-z1.pdb, discussed elsewhere herein) ⁇ a root mean square deviation from the backbone atoms of the amino acids of such protein of not more than 1.5 A.
  • An illustrative embodiment of this aspect of the invention is a conventional 3.5" diskette, DAT tape or hard drive encoded with a data set, preferably in PDB format, comprising the coordinates of Appendix I, Appendix II or Appendix III.
  • the machine-readable data storage medium comprises a data storage material encoded with a first set of machine readable data which comprises the Fourier transform of the structural coordinates set forth in Appendix I, Appendix II or Appendix III (or again, a derivative thereof), and which, when using a machine programmed with instructions for using said data, can be combined with a second set of machine readable data comprising the X-ray diffraction pattern of a molecule or molecular complex to determine at least a portion of the structural coordinates corresponding to the second set of machine readable data.
  • FIG. 8 illustrates one version of these embodiments.
  • the depicted system includes a computer A comprising a central processing unit (“CPU"), a working memory which may be, e.g., RAM (random-access memory) or “core” memory, mass storage memory (such as one or more disk drives or CD-ROM drives), one or more cathode-ray tube (“CRT”) display terminals, one or
  • CPU central processing unit
  • working memory which may be, e.g., RAM (random-access memory) or “core” memory
  • mass storage memory such as one or more disk drives or CD-ROM drives
  • CRT cathode-ray tube
  • keyboards one or more input lines (IP), and one or more output lines (OP), all of which are interconnected by a conventional bidirectional system bus.
  • IP input lines
  • OP output lines
  • Input hardware B coupled to computer A by input lines, may be implemented in a variety of ways.
  • Machine-readable data of this invention may be inputted via the use of a modem or modems connected by a telephone line or dedicated data line JL.
  • the input hardware may comprise CD-ROM drives or disk drives D_.
  • a keyboard may also be used as an input device.
  • Output hardware coupled to computer A by output lines, may similarly be implemented by conventional devices.
  • output hardware may include a CRT display terminal for displaying a graphical representation of a protein of this invention (or portion thereof) using a program such as QUANTA as described herein.
  • Output hardware might also include a printer, so that hard copy output may be produced, or a disk drive, to store system output for later use.
  • the CPU coordinates the use of the various input and output devices, coordinates data accesses from mass storage and accesses to and from working memory, and determines the sequence of data processing steps.
  • a number of programs may be used to process the machine- readable data of this invention. Examples of such programs are discussed in reference to the computational methods of drug discovery as described herein. Specific references to components of the hardware system of FIG. 8 are included as appropriate throughout the following description of the data storage medium.
  • FIG. 9A shows a cross section of a magnetic data storage medium 100 which can be encoded with a machine-readable data that can be carried out by a system such as a system of FIG. 8.
  • Medium 100 can be a conventional floppy diskette or hard disk, having a suitable substrate 101 , which may be conventional, and a suitable coating 102, which may be conventional, on one or both sides, containing magnetic domains (not visible) whose polarity or orientation can be altered magnetically.
  • Medium 100 may also have an opening (not shown) for receiving the spindle of a disk drive or other data storage device 24.
  • the magnetic domains of coating 102 of medium 100 are polarized or oriented so as to encode in a manner which may be conventional, machine readable data such as that described herein, for execution by a system such as a system of FIG. 8.
  • FIG. 9B shows a cross section of an optically-readable data storage medium 110 which also can be encoded with such machine-readable data, or set of instructions, which can be carried out by a system such as a system of FIG. 8.
  • Medium 110 can be a conventional compact disk read only
  • Medium 100 preferably has a suitable substrate 111 , which may be conventional, and a suitable coating 112, which may be conventional, usually of one side of substrate 111.
  • coating 112 is reflective and is impressed with a plurality of pits 113 to encode the machine-readable data.
  • the arrangement of pits is read by reflecting laser light off the surface of coating 112.
  • a protective coating 114 which preferably is substantially transparent, is provided on top of coating 112.
  • coating 112 has no pits 113, but has a plurality of magnetic domains whose polarity or orientation can be changed magnetically when heated above a certain temperature, as by a laser (not shown).
  • the orientation of the domains can be read by measuring the polarization of laser light reflected from coating 112.
  • the arrangement of the domains encodes the data as described above.
  • the first 259 residue segment of ZAP-70 consists of two SH2 domains that are connected by a helical region.
  • the overall fold is Y-shaped where the SH2 domains constitute each upper branch and the intervening 65 amino acid domain forms the stem.
  • the fragment of ZAP-70 used in the crystal structure determination terminates before the kinase domain; therefore, we refer to the two SH2 regions as ZAP-N and ZAP-C, for ZAP N-terminal SH2 and ZAP C- terminal SH2, respectively.
  • Each of the individual ZAP SH2 domains possesses a central antiparallel ⁇ -sheet that is flanked by two ⁇ -helices.
  • the inter-SH2 region begins as a ⁇ -strand that is a continuation of the central sheet of ZAP-N. This is followed by two antiparallel ⁇ -helices that intertwine to form a coiied-coil motif. The two
  • the i peptide is extended over both faces of the SH2 domains, straddles both central ⁇ -sheets, and makes extensive contacts with the protein surface.
  • the binding orientation is head to tail, that is, the N-terminus of the peptide is in contact with the C-terminal SH2 domain.
  • the N- terminal pYXXL segment of the peptide is bound to ZAP-C in a conformation similar to that seen for singiy-phosphorylated peptides bound to isolated SH2 domains. 20, 21
  • the peptide segment that separates the two pYXXL motifs is largely in contact with ZAP-C.
  • the C-terminal phosphotyrosine is bound in -a pocket that is formed by contributions from both SH2 domains.
  • the remainder of the second pYXXL motif is bound in a fashion similar to the first motif and in other complexes. 20 ' 21
  • helix A is longer by three residues, which extends the helix nearly a full turn.
  • the C- terminal SH2 domain of ZAP also possesses minor extensions to several secondary structural elements. Strands B, C, and E are longer by one or two residues. Helix A is also extended by a single residue. Strand F consists of only two residues and replaces the FB loop. The BC loop of ZAP-C is extended.
  • the inter-SH2 spacer begins in a type II reverse turn followed by a long ⁇ -strand that makes significant contact to strand A of ZAP-N, thus forming an extension to the central ⁇ sheet.
  • Hydrogen bonds exist between main chain atoms of Gin 111 and Tyr 12 and between Gin 111 and Ser 14.
  • a water-mediated contact exists between the main chain atoms of Glu 109 and Tyr 12.
  • hydrophobic packing exists between Leu 108 and Tyr 12 and between Pro 110 and Phe 11.
  • helix C a five-turn ⁇ -helix
  • helix D a fifth ⁇ -helix
  • helix D curves around the axis formed by helix C.
  • Helix D is distorted, with a break at Pro 147; a second break occurs at Ala 154 which precedes a short 3 ⁇ o-helix that spans residues Thr 155 - Met 161.
  • Helices C and D both make several hydrophobic contacts to ZAP-C, most notably a p-stacked arrangement of Phe 115 ( ⁇ C) to Trp 233.
  • Several water-mediated hydrogen bonds exist between helix D and ZAP- C. These antiparallel helices form a coiled-coil structure, with direct contact between several hydrophobic residues forming its core.
  • the region separating the two domains is highly variable in length. This region may be as short as 10-15 residues (e.g., PLC- ⁇ 1 , SHPTP-1 and -2), which would force the two SH2 domains into a back-to-back orientation.
  • SYK has an inter-SH2 domain region which is of comparable length to that of ZAP, exhibits 68% sequence identity to the helical spacer described here. It should maintain the same overall conformation observed in ZAP-NC.
  • tandem SH2 domains of the p85 subunit of phosphtidylinositol 3' kinase are connected by a significantly larger domain of 163 residues which has been predicted to also form a coiled coil of two antiparallel ⁇ -helices. 22
  • the ZAP-NC: ⁇ complex includes a 19 amino acid peptide that is phosphorylated on both tyrosine residues and is based on the first ITAM-containing segment of the human TCR ⁇ chain ( ⁇ i ) which has the sequence NQLpYNELNLGRREEpYDVLD [SEQ ID NO 14] .
  • ⁇ i the first ITAM-containing segment of the human TCR ⁇ chain
  • NQLpYNELNLGRREEpYDVLD SEQ ID NO 14
  • numbering for peptide residues begins at ⁇ Asn 1.
  • the bound conformation of the ⁇ -j peptide is largely extended, although nearly one full ⁇ -helical turn exists between residues ⁇ Asn 8 and ⁇
  • Binding of Motif-1 (-pYNEL-) is Exclusive to ZAP-C
  • the amino terminal pYXXL motif of ⁇ 1 is associated exclusively with ZAP-C.
  • the first two residues of ⁇ , ⁇ Asn 1 and ⁇ Gin 2 are largely involved in intrapeptide interactions.
  • the single contact between ⁇ Leu 3 and ZAP-NC, a hydrogen bond between the main chain carbonyl of ⁇ Leu 3 and NH1 of Arg 170, is typical for the pY-1 residue.
  • the pocket for ⁇ pTyr 4 is formed by residues from helix A, strands B, C, and D, and the BC loop. Hydrophobic contacts involve residues from ⁇ D, from which His 210, Tyr 211 , and Leu 212 form one edge of the pTyr cavity. In addition, ⁇ Asn 1 and ⁇ Gin 2 of the peptide itself form hydrophobic contacts on the opposing side. The side chain of Leu 212 is twisted away from the pTyr ring and is packed against Trp 131 from a symmetry-related molecule. This neighboring Trp, which constitutes the only intermolecular crystal contact with any ⁇ * ) residue, is also in hydrophobic contact to ⁇ pTyr 4.
  • Direct hydrogen-bonding contacts to the phosphate are made by only three residues. Arg 170 ( ⁇ A) and Arg 190 ( ⁇ B) interact through their terminal nitrogens. Arg 192 is the only residue in the BC loop of sufficient length for direct hydrogen bonding to the phosphate group and interacts via its N ⁇ . The BC loop is extended; thus, the pTyr binding region resembles a deep groove that continues toward the AA loop. The inclusion of the pY-2 and pY-3 residues as an integral part of the binding site results in the formation of a channel into which the pTyr protrudes. Five waters with very strong density and low temperature factors exist in this region and are part of a large hydrogen-bonding network.
  • the pY+1 and pY+2 residues are extended along the surface of the protein.
  • the pY+1 residue ( ⁇ Asn 5) makes contacts that are similar to those observed in the hamster middle T peptide (...pYEEl%) in complex with the SH2 domains of Lck 20 and v-Src. 21
  • the pY+2 residue ( ⁇ Glu 6) is directed away from the surface of the protein.
  • the pocket that surrounds ⁇ Leu 7 (pY+3) is very deep and is formed by residues from ⁇ D, the EF loop, helix B and the BG loop. Due to the size of this pocket, ⁇ Leu 7 is directly contacted by only 5 residues - Tyr 211 , lie 223, Gly 226, Gly 245, and Leu 246. The depth of this pocket is partially due to the presence of a leucine in helix B in ZAP-C that is occupied by a tyrosine in many other SH2 domains. 23 Even in the absence of tyrosine, very strong density is observed for two waters near this site. The main chain of ⁇ Leu 7 is involved in a water-mediated hydrogen bond to the carbonyl oxygen of Pro 224. A second contribution to the overall shape of the pY+3 pocket is provided by a repositioning of the ⁇ turn in the EF loop. In complexes of isolated SH2 domains, this loop is involved in forming the steep solvent-exposed wail of the
  • consists of seven amino acids which make the bulk of their contacts to ZAP-C. Since nearly a full turn of an ⁇ -helix begins at ⁇ Asn 8 and continues to ⁇ Arg 12, many contacts for this sequence are intrapeptide.
  • ⁇ Asn 8 makes a main chain hydrogen bond to the carbonyl oxygen of Gly 245 (BG), and ⁇ Arg 12 is involved in both direct and water-mediated hydrogen bonds to the backbone carbonyl of Glu 225 (EF loop); two other water-mediated hydrogen bonds connect ⁇ Arg 12 to Gly 226 and Lys 228.
  • the side chains of ⁇ Leu 9 and ⁇ Arg 12 close off the pY+3 pocket of ZAP-C.
  • ⁇ Glu 13 makes a main chain hydrogen bond to the backbone carbonyl of Asp 244.
  • ⁇ Glu 13 is involved in a direct hydrogen bond through its side chain carboxyl to the side chain amino group of Lys 242 (ZAP-C ⁇ B) which is an integral part of the phosphotyrosine pocket of the N-terminal SH2 domain, ⁇ Glu 13 also maintains van der Waals contact to Lys 242, as well as to Tyr 238 (ZAP-C ⁇ B) and the guanidinium group of Arg 17 (ZAP-N ⁇ A), which also contribute to the N-terminal pY pocket, ⁇ Glu 14 maintains the characteristic pY-1 main chain carbonyl hydrogen bond to both terminal nitrogens of Arg 17 which, in turn, contacts the aromatic ring and phosphate group of ⁇ pTyr 15.
  • the side chain of Arg 17 is positioned over the aromatic ring of ⁇ pTyr 15, forming an amino-aromatic contact in addition to bridging the carbonyl of the pY-1 residue to the phosphate oxygens of ⁇ pTyr 15.
  • the phosphate group is closely associated with the side chains of Tyr 238 (ZAP-C ⁇ B), Lys 242 (ZAP-C ⁇ B), Arg 17 ( ⁇ A), and Arg 37 ( ⁇ B), forming a total of six direct hydrogen bonds.
  • Six water-mediated hydrogen bonds exist between the phosphate group and Arg 17 ( ⁇ A), Cys 39 ( ⁇ C), Leu 40 (BC loop), Arg 41 (BC loop), and Lys 242 (ZAP-C ⁇ B).
  • each oxygen of the phosphate group possesses its full complement of hydrogen-bonding partners.
  • the pY+1 and pY+2 residues make contacts that are characteristic for these positions in other SH2 complexes.
  • 20 ' 21 • 23 ⁇ Leu 18 resides in a hydrophobic pocket that is of similar dimension to the pY+3 pockets observed in high affinity peptide complexes with Src family SH2 domains.
  • a single water- mediated hydrogen bond connects the main chain NH of ⁇ Leu 18 to the carbonyl of Ala 72 on the EF loop.
  • ⁇ D contributes Phe 59; interaction with the EF loop involves Ile 71 , Ala 72, Gly 73, and Gly 74; helix B presents Tyr 87; and the
  • BG loop makes contact via Gly 93 and Leu 94.
  • resides in weaker density and appears to form only one hydrogen bond from its main chain nitrogen to the carbonyl of Gly 93 (BG loop).
  • the total interaction area between the ZAP-N and ZAP-C SH2 domains is small, measuring only ca. 200 A 2 .
  • the surface area of ZAP-N that is buried by ZAP-C and the inter-SH2 spacer is only about 400 A 2 ; the corresponding buried area in ZAP-C is not significantly larger.
  • Total burial of ZAP-N in the full complex is 620 A 2 which accounts for approximately 13% of the total surface area of the domain. Conversely, burial of
  • ZAP-C is computed to be ca. 1000 A 2 which constitutes 20% of its total surface area. This difference is due to the presence of a large solvent accessible channel formed by the convergence of the convex side of the BC loop of ZAP-N, the FB loop and helix B of ZAP-C, and both helices of the inter-SH2 spacer.
  • This irregularly shaped funnel has an approximate diameter of 5-7 A, and extends fully enclosed for ca. 12 A before flaring open for an additional 8 A.
  • each SH2 domain contributes nine residues. Most of the contacts are through hydrogen bonds, and most of these are water-mediated. However, some van der Waals contacts do exist.
  • the inter-SH2 spacer is likely to stabilize the appropriate orientation for tandem binding by permitting only minor displacements through scissoring or wagging motions.
  • uncomplexed ZAP-NC exists as multiple bands which collapse into a single band when the ⁇ -
  • the overall backbone root-mean-square (r.m.s.) deviation is 1.07A ; the same measurement for ZAP-N or ZAP-C to Src family SH2 domains (individually) is typically 1.50 A, although the percentage of sequence identity is similar.
  • the loop regions display the largest positional variance, most notably loops AA, BC, CD, and EF.
  • the CD loops of both ZAP-N and ZAP-C have a large truncation relative to the SH2 domains of the Src famiiy; this truncation is also evident from the sequences of a large number SH2 domains.
  • the pY+3 pocket of ZAP-C is strikingly large compared to this site in other SH2 domains. This is due, in part, to repositioning of ZAP-C EF residues Pro 224 and Glu 225, as described earlier. Aside from the absence of a Tyr in ⁇ B, the location of other side chains that form this pocket are notably similar to the corresponding sites in Lck and Src.
  • the tandem SH2 domains of ZAP-70 exhibit strong selectivity for the phosphorylated ⁇ and ⁇ subunits of the TCR, while isolated SH2 domains from other proteins bind more promiscuously to many tyrosine phosphorylated proteins in total cell extracts.
  • ° Ligand binding and selectivity for isolated SH2 domains is mediated by recognition of a phosphotyrosine and several residues C-terminai to phosphotyrosine, particularly the hydrophobic residue at the pY+3 position 1 2 .
  • the high degree of selectivity of ZAP-70 for doubly-phosphorylated ITAM sequences appears to be a consequence of multiple structural features.
  • the distance between the two pYXXL motifs of the ⁇ or ⁇ chain provides properly spaced partners for a pair of SH2 domains that are tethered in close association by an inter-SH2 coiled coil. Association of the pair of SH2 domains with the phosphotyrosines and other ITAM residues stabilizes a conformation that permits direct interaction between the domains and hence the formation of a deep pocket for sequestering one phosphotyrosine at the domain interface.
  • ZAP-NC exhibits high affinity for doubly-phosphorylated ITAMS and selectivity for the ⁇ and ⁇ chains
  • the individual SH2 domains of ZAP-70 have not been found to bind appreciably to phosphorylated peptides 5 .
  • ZAP-NC binds to monophosphorylated ITAM-based peptides with affinities that are 100-1000 times Iower than for the corresponding doubly- phosphorylated ones. 17"1 9 ' 25 Consequently, for high-affinity binding, both SH2 domains must cooperate, and two phosphotyrosine residues must be present and arranged appropriately.
  • the structural manifestation of this cooperativity and selectivity is also the most remarkable feature of the complex between ZAP-NC and ⁇ i , that is, the convergence of residues from both SH2 domains to enmesh pTyr 15.
  • SH2 domains adopt their native fold when extracted from their natural molecular context and possess their full ability to recognize and bind to phosphorylated proteins. 12, 14
  • N-terminai SH2 domain of ZAP-70 if expressed in isolation, is incomplete.
  • the groove-like nature of the pY pocket of ZAP-C suggests that this domain may also require contributions from neighboring domains or proteins.
  • the inter-SH2 region constrains the SH2 domains within a distance that permits association. However, because a significant portion of the antiparallel helices are directed away from the SH2 domains, this region may also be involved in inter- or intramolecular contacts, regulation of kinase activity, and/or receptor clustering. The evidence that this domain forms a coiled coil of ⁇ -helices is of great interest, since these structural units are commonly involved in protein-protein interactions. 27
  • the inter-SH2 region of the p85 subunit of PI 3-K which has been predicted to form a coiled coil, is necessary and sufficient for interaction of p85 with the p1 10 catalytic subunit of PI 3-K.
  • ZAP interdomain is involved in regulation of its kinase activity.
  • the interdomain may inhibit catalytic activity directly or indirectly, and this inhibition might be relieved upon binding of the SH2 domains to the ITAM.
  • PI 3-K 25 and SYK 28 the PTK homologous to ZAP-70 , support such a. model.
  • phosphorylated ITAM peptides derived from the ⁇ subunit of the IgE receptor increase SYK kinase activity by 5-10 fold.
  • Another function for the inter-SH2 region may be to bind to proteins that either regulate ZAP-70 activity, such as Lck and/or Fyn, or that are substrates for ZAP-70.
  • Tyrosine 126 in the inter-SH2 region is phosphorylated by Lck in vitro 32 and could be involved in interactions with other SH2 domain-containing proteins.
  • the inter-SH2 domain may be important for the intermolecular association between ZAP-70 molecules which might occur in the activated TCR complex.
  • SYK should also exhibit these structural features in view of its functional similarities and sequence identity of 57% with respect to ZAP. SYK is expressed in several types of
  • ZAP-70 has emerged as an attractive target for the development of safe and potent immunosuppressive drugs.
  • ZAP-70 has been shown to be required for T cell-mediated immune responses in humans, and loss of ZAP-70 does not affect other tissues. 8
  • ZAP antagonists would specifically inhibit T cells and avoid the toxicity of the currently used immunosuppressive drugs, FK506 and cyclosporin 35 ' 36 , which target the more ubiquitously expressed protein calcineurin.
  • FK506 and cyclosporin 35 ' 36 which target the more ubiquitously expressed protein calcineurin.
  • 37 ' 38 This protein phosphatase is required for T cell immune responses, as well as functions in several other tissues, and as a consequence, cyclosporin and FK506 cause serious side effects in the kidney and central nervous system which limit their application largely to pateients with organ transplant rejection. 36
  • New immunosuppressive drugs with less toxicity are needed to expand the routine use of such therapies to autoimmune diseases.
  • One approach to inhibition of T cell activation is to develop small (i.e., preferably having a molecular weight below about 1200, more preferably below about 750 and even more preferably below about 500), preferably non-peptidic, membrane permeant, molecules that bind to ZAP and prevent its association with the TCR.
  • small i.e., preferably having a molecular weight below about 1200, more preferably below about 750 and even more preferably below about 500
  • a compound may bind, preferably with high affinity, to either SH2 domain of ZAP-70 or to the inter-SH2 domain interaction.
  • Our crystal structure reveals the molecular details of the three dimensional structure of ZAP and provides insights into its interactions with the TCR.
  • the unique structural features of each SH2 ligand binding site and the unanticipated inter-SH2 association now can be exploited for structure-based design of highly specific small molecule ZAP ligands and structurally biased compound libraries.
  • - 33 - protein currently considered to be most closely related to ZAP is SYK. (see figure of sequence alignment).
  • ZAP-NC Using the structure of ZAP-NC, a three-dimensional model of Syk-NC can be obtained through homology modeling. Prior to solving the ZAP-NC structure, this would have been difficult if not impossible since the sequence identity between the SH2 domains of Syk and SH2 domains of known structure is low and none of the previously solved SH2 domains contain two SH2 domains.
  • Other currently known proteins with tandem SH2 domains are PLC ⁇ , PI3K, rasGAP, SH-PTP1 , and SH-PTP2. Additional proteins with two SH2 domains are expected to be discovered through genome sequencing or other cloning methods.
  • ZAP-NC protein tyrosine kinase ZAP-70
  • Structure-based approaches include de Novo molecular design, computer-aided optimization of lead molecules, and computer-based selection of candidate drug structures based on structural criteria.
  • New peptidomimetic modules may be developed directly from the structure of the peptide ligand by design or database searches for conformationally-restricted peptide replacements.
  • structure-based lead discovery may be accomplished using the target protein structure stripped of its ligand. Multiple uncomplexed states of ZAP-NC can be generated by several methods to provide additional target conformations.
  • the experimental coordinates and the resulting uncomplexed models can be subjected to techniques such as receptor site mapping to identify sites of favorable interaction energies between the structure of the target protein and potential ligands or chemical moieties ("fragments" or "seeds"). Such evaluation may be followed by procedures such as fragment seed linking and growth.
  • Fragment seed linking refers to methods for designing structures that contain "linked” "seeds", i.e. chemical structures comprising two or more of the mapped moieties appropriately spaced to reach the respective sites of favorable interactions.
  • Growth refers to the design of structures which extend, based on receptor site mappping or to fill available space, a given molecule or moiety. Based on the receptor site mapping data, one may also select potential ligands from databases of chemical structures.
  • the structure of ZAP-NC permits one to generate a high-quality model of Syk-NC by either knowledge-based homology template methods or iterative site-mutations followed by minimizations.
  • the generated structure of Syk-NC may
  • Peptidomimetics of the ⁇ -j peptide may be developed from the bound conformation of a peptide ligand by design, by searching databases for replacements of one or more peptide segments, or by enhancement of existing ligand-protein interactions (i.e., by replacing a component moiety of a ligand with a substitute moiety capable of greater interaction with the target protein, whether through accessible protein contact points or by extrusion of otherwise sequestered waters).
  • Knowledge of the bound conformation of a peptide can suggest avenues for conformational restriction and peptide bond replacement.
  • a less biased approach involves computer algorithms for searching databases of three dimensional structures to identify replacements for one or more portions of the peptide ligand, preferably non-peptidic replacement moieties.
  • Programs that may be used to synchronize the geometric and steric requirements in a search applied to ZAP-NC include CAVEAT (University of California, Berkeley), HOOK (MSI), and ALADDIN (Daylight Software). All of these searching protocols may be used in conjunction with existing corporate databases, the Cambridge Structural Database, or available chemical databases from chemical suppliers.
  • the incorporation into a ligand structure of hydrogen-bond donating or accepting groups that can displace ordered water molecules usually provides a significant entropic gain that leads to a favorable free energy of binding.
  • ordered waters are identifiable from the structure, and other ordered waters may be located during computer simulations of a fully solvated structure, as described more thoroughly in a subsequent section.
  • the target protein may be desired in view of the flexibility of the phosphotyrosine binding region, the nature of the interface between the two SH2 domains, and overall, in view of the possibility of an induced fit, i.e., conformational changes in both ligand and protein upon binding.
  • the loop that connects ⁇ -strands B and C has been reported to act as a functional hinge in Src-family SH2 domains.
  • charged residues in the phosphotyrosine binding pocket are capable of side chain reorientations.
  • - 35 - methods such as Metropolis Monte Carlo or molecular dynamics simulations (implemented in programs such as MCPro [Yale Univeristy], AMBER [UCSF], CHARMm [Harvard University], and GROMOS [ETH/Groningen]) may be used locally to generate Boltzmann distributions of uncomplexed states, and hence provide a set of additional conformations that are valid for molecular design. Alternate side chain reorientations can also be examined by Dead End Elimination and A * algorithms (University of Southhampton), by iterative systematic conformational searches of each side chain, or by comparison of each residue type to members of the Protein Data Bank that have the same backbone torsions.
  • Metropolis Monte Carlo or molecular dynamics simulations may be used locally to generate Boltzmann distributions of uncomplexed states, and hence provide a set of additional conformations that are valid for molecular design. Alternate side chain reorientations can also be examined by Dead End Elimination and A * algorithms (Univers
  • Valid conformations of the BC loop may be created by searching the Brookhaven Protein Data Bank for loops that have similar anchoring geometries or by imposing random backbone conformations within the selected loops and filtering the results to fit the anchor residues. Both of these knowledge-based methods generate initial structures which can be subjected to force-field minimizations to produce feasible geometries.
  • SH2 domains provides opportunities for exploring additional conformational states.
  • the interface that is exclusive to the two SH2 domains provides a total buried surface area of only about 200 A 2 , and consists largely of hydrogen bonding contacts, many of which are mediated by water.
  • isoelectric focusing gels suggest that uncomplexed ZAP-NC exhibits conformational mobility between the two SH2 domains that is subdued upon binding to the ⁇ -
  • Molecular dynamics simulations of a fully solvated ZAP-NC may provide insight into the structural manifestation of a possible dissociation between the SH2 domains, and an additional target conformation of the uncomplexed protein.
  • Receptor site mapping encompasses a variety of computational procedures that identify energetically favorable binding sites on macromolecules.
  • the most straighforward procedures involve "painting" a solvent-accessible surface (or an otherwise generated cast) of the macromolecular target according to empirically determined physical properties, such as electrostatic or lipophilic potential, degree of curvature, and hydrogen-bonding character.
  • Such methods for thus characterizing the surface of a macromolecule are incorporated in programs such as Grasp (Columbia University), DelPhi (Biosym Technologies), MOLCAD (Tripos, Inc.), and Hint (Virginia Commonwealth University).
  • Subsequent molecule design involves identification or design of ligands that possess features complimentary to the identified surface characteristics. More advanced algorithms involve the actual calculation of interaction enthalpies between the target and potential ligands or fragments. In practice, the coordinates of
  • the protein or protein fragment of interest (which may be rotated or otherwise transformed) are stripped of any undesired ligand (or portion thereof) and/or of any undesired solvent molecules.
  • the coordinates are then processed to attach molecular mechanics parameters to the atomic positions to provide a processed target for mapping.
  • the target may be partitioned into discrete binding sites.
  • the target or partitioned sites thereof are flooded with given functional group fragments that are subsequently allowed to relax into desired locations, as in the program MCSS (Molecular Simulations, Inc.), or are encased within a regular lattice of site points on which single fragment probes are positioned sequentially; examples of programs that exploit the site-lattice algorithm include Grin/Grid (Molecular Discovery, Ltd.), Ludi (Biosym Technologies), Leapfrog (Tripos, Inc.), and Legend (University of Tokyo). In both techniques, the enthalpic contribution to binding affinity is estimated with a molecular mechanics force- field, and appropriate positions of selected functional groups are determined systematically.
  • MCSS Molecular Simulations, Inc.
  • a box is defined enclosing a desired portion of the target within a defined lattice.
  • the lattice resolution i.e., the distance between lattice points, may be defined by the practitioner or may be set by the computer program.
  • other parameters of points within the lattice such as hydrophobicity or other characteristics, may be similarly defined.
  • Probes i.e. computer models of one or more selected moieties, functional groups, molecules or molecular fragments are positioned at lattice points and the interaction energy of the probe- target pair is determined for each such lattice point.
  • the data for each selected moiety, functional group, etc. is collected and may be recovered as a data set, visualized on a computer monitor or printed out in various text or graphic formats.
  • the model is then subjected to group minimization (i.e., molecular mechanics minimization) calculations to identify points or areas of favorable interaction. Data may be handled as in the lattice approach.
  • Receptor site mapping may be applied to the 3-D structure of a ZAP family member to design or select ligands capable of binding to an SH2 domain or other site within the ZAP-NC (or ZAP family-NC) structure.
  • Receptor site maps provide the seeds for ligand evolution via Database searches, which are described above, and for Grow/link methods for de Novo design of new chemical entities.
  • Programs for ligand growth first access extensible fragment dictionaries in order to place appropriate functional groups at site points. A genetic algorithm or a subgraph isomorphism protocol is then invoked to connect the fragments with small aliphatic chains or rings. Stochastic enhancements may be introduced by modification of internal degrees of freedom as well as translation and rotation of the candidate model within the binding cavity. The resulting sets of molecules are scored and filtered by functions that consider the steric constraints of the binding site, the complimentarity of electrostatic and hydrophobic interactions, and a solvation estimate. Programs of this type that couid be applied for the design of new ligands for ZAP-NC include Ludi (Biosym Technologies), Leapfrog (Tripos, Inc.), Legend (University of Tokyo),
  • DOCK Universally of California, San Francisco
  • similar programs fill a given binding site with the smallest set of atom-sized spheres possible; a database search then attempts to orient ligands such that the atoms superimpose onto the centers (or "nuclei") of the site-filling spheres.
  • the shape complimentarity is augmented by scoring functions that include the steric requirements of the cavity and a potential energy function.
  • optimization of ligands may be enhanced using the three dimensional structural of ZAP-NC.
  • Use of receptor site maps or hydropathic profiles of ZAP-NC may be used to identify preferred positions for functional group components of ligands. and can be used to filter or constrain conformational searches of ligand structures which would otherwise typically be controlled by minimal steric considerations of the iigand structures themselves.
  • the availability of an explicit binding site also permits one to determine the mode of ligand binding to the target protein via methods that utilize force-fields directly in simulated annealing, distance geometry, Metropolis Monte Carlo, or stochastic searches for binding modes.
  • Examples of programs that can be applied to rationalize ligand binding to ZAP-NC include Autodock (Scripps Clinic), DGEOM (QCPE #590), Sculpt (Interactive Simulations, Inc.), or any of the molecular dynamics programs described above.
  • Autodock Scripps Clinic
  • DGEOM QCPE #590
  • Sculpt Interactive Simulations, Inc.
  • any of the molecular dynamics programs described above Once a tractable set of possible binding orientations is obtained, one can readily identify the appropriate mode of binding through modifications in test ligands designed to alter in a predictable fashion the binding affinity of each model under consideration. For instance, a ligand may be modified to contain a functional group at a position which is inconsistent with one binding model, yet consistent with another model. Binding data can then be used to weed out "disproven” models.
  • An alternative protocol for ligand optimization involves 3D database searching in conjunction with knowledge of the binding site. Modeling can reveal multiple candidates for the bioactive conformation of a given ligand. A probe for the correct conformation can include a 3D search to identify several constrained mimics of each possible conformer. Structure-activity relationships of the unconstrained ligand would suggest which functional groups should be retained in the constrained mimics. Finally, the steric and electrostatic requirements of the binding site could constitute a filter for prioritizing the resultant possibilities.
  • the structure of ZAP-NC permits accurate model-building of homologous proteins, and their subsequent use in drug design.
  • the SH2 domains and inter-domain coiled coil regions of the protein tyrosine kinase p72 s >* share a high degree of sequence similarity with ZAP-70.
  • the ZAP-NC structure may be readily used in the development of a reliable model of Syk-NC by either knowlege-based template building methods, or by iterations of directed point mutations followed by local molecular mechanics minimizations. Examples of programs that can be applied in the development of a model of Syk-NC include Composer (Birbeck College), Modeler (MSI), and Homology (Biosym Technologies). The resultant model can then be subjected to any of the CADD techniques described above.
  • SPR methodologies measure the interaction between two or more macromolecules in real-time through the generation of a quantum-mechanical surface plasmon.
  • the SPR methodology as exploited by the BIAcore Biosensor ® (Pharmacia Biosensor, Piscatawy, NJ) focuses a beam of polychromatic light at the interface between a gold film (provided as a disposable biosensor "chip") and a buffer compartment that can be regulated by the user.
  • Attached to the gold film is a 100 nm thick "hydrogel” composed of carboxylated dextran which provides a matrix for the
  • the interaction between the two components can be measured in real time based on the accumulation of mass in the evanescent field and its corresponding effects of the plasmon resonance as measured by the depletion spectrum.
  • This system permits rapid and exremely sensitive real-time measurement of the molecular interactions without the need to label either component.
  • Fluorescence polarization is a measurement technique that can readily be applied to protein-protein and protein-iigand interactions in order to derive IC50s and K-js of the association reaction between two molecules.
  • one of the molecules of interest must be conjugated with a fluorophore: this is generally the smaller molecule in the system (in the case of a SH2 system, a phospho-tyrosine-containing peptide).
  • the sample mixture containing both the iigand-probe conjugate and the protein receptor, is excited with vertically polarized light. Light is absorbed by the probe fluorophores, and re-emitted a short time later. The degree of polarization of the emitted light is measured. Polarization of the emitted light is dependent on several factors, but most importantly on viscosity of the solution and on the apparent molecular weight of the fluorophore.
  • Fluorescence polarization equilibrium binding assays have been adapted for ZAP, Syk, and Src domains.
  • a binding curve of a doubly-phosphorylated ⁇ -1 sequence to N.C-ZAP, with associated Scatchard plot of the data is shown in FIG. 5.
  • a compound preferentially inhibits the interaction of a particular SH2-containing protein with its natural ligand (or a portion thereof or analog based thereon), e.g. at least an order of magnitude, and even more preferably, at least two orders of magnitude better (by any measure) than it inhibits some other SH2-ligand interaction.
  • Such compounds may be further evaluated for activity in inhibiting cellular or other biological events mediated by a pathway involving the interaction of interest using a suitable cell-based assay or an animal model.
  • Cell-based assays and animal models suitable for evaluating inhibitory actvity of a compound with respect to a wide variety of cellular and other biological events are known in the art. New assays and models are regularly developed and reported in the scientific literature.
  • compounds which bind to ZAP-70 may be evaluated for biological activity in inhibiting T ceil activation using any conventional assay methods and materials.
  • compounds which bind to ZAP may be assayed for inhibition of CD4+ and CD8+ T-lymphocytes in vitro and for lack of in vitro toxicity on cytotoxic T-cells within the dose range used to demonstrate in vitro activity.
  • a battery of in vivo models may be used to profile the breadth of the compound's immunosuppressive activity and compare the profile to those of positive controls such as cyclosporin and FK506. Comparisons may also be made to other currently accepted immunosuppressive compounds, i.e. rapamycin, cyclophosphamide, and leflunomide.
  • Initial in vivo screening models include: Delayed type hypersensitivity testing, Allogeneic skin transplantation, and Popliteal lymph node hyperplasia. Compounds demonstrating optimal profiles in the above models are advanced into more sophisticated models designed to confirm immunosuppressive activity in specific therapeutic areas including: Rheumatoid arthritis, Transplantation, Graft vs. host disease, and Asthma.
  • Compounds which bind to SYK may be evaluated for inhibitory activity in a mast cell or basophil degranulation assay.
  • the inhibitory activity of a compound of this invention with respect to cellular release of specific mediators such as histamine, ieukotrienes, hormonal mediators and/or cytokines as well as its biological activity with respect to the levels of phosphatidylinositol hydrolysis or tyrosine phosphorylation can be characterized with conventional in vitro assays as an indication of biological activity.
  • mediators such as histamine, ieukotrienes, hormonal mediators and/or cytokines
  • conventional in vitro assays as an indication of biological activity.
  • active compounds such as leflunomide (and its active metabolite, A771726), vanadate, staurosporine, genistein, or other compounds, including clinically relevant compounds, which can be used as positive controls.
  • Compounds which bind to SYK may also be tested in an ex vivo assay for their ability to block antigen-stimuiated contraction of sensitized guinea pig tracheal strip tissue. Activity in this assay has been shown to be useful in predicting the efficacy of potential anti-asthma drugs. Numerous animal models of asthma have been developed and can be used (for reviews, see
  • compounds which bind to an SH2-bearing protein involved in the transduction of a signal involved in the initiation, maintenance or spread of cancerous growth may be evaluated in relevant conventional in vitro and in vivo assays. See e.g., Ishii et al., J. Antibiot. XLIM 877-1878 (1989) (in vitro evaluation of cytotoxic/antitumor activity); Sun et al, US Patent 5,206,249 (issued 27 April 1993)(in vitro evaluation of growth inhibitory activity on cultured leukemia cells); and Sun et al, supra (xenograft models using various human tumor cell lines xenografted into mice, as well as various transgenic animal models).
  • Single and multiple (e.g., 5 to 7 days) dose investigative toxicology studies are typically performed in the efficacy test species using the intended route of administration for the efficacy study. These investigative toxicology studies are performed to identify maximum tolerated dose, subjective bioavailability from the intraperitoneal or oral routes of administration , and estimation of an initial safety margin, initial bioavailability and pharmacokinetics (blood clearance) of the compounds may be determined, with standard cold or radioactive assay methods, to assist in defining appropriate dosing regimens for the compounds in the animal models.
  • Acidic residues from that region which are capable of entering hydrogen-bonding interactions or ionic (sait-bridge) interactions with moieties on ligand molecules are listed in Table B.
  • Basic residues from that region which are capable of entering hydrogen-bonding interactions or ionic (salt-bridge) interactions with moieties on ligand molecules are listed in Table C.
  • Neutral residues from that region which are capable of entering hydrogen-bonding interactions with moieties on ligand molecules are listed in Table D.
  • Residues from that region having appropriately disposed backbone amide carbonyls which are capable of entering hydrogen-bond accepting interactions with moieties on ligand molecules are listed in Table E.
  • Residues from that region having appropriately disposed backbone amide nitrogens which are capable of entering hydrogen-bond donating interactions with moieties on iigand molecules are listed in Table F.
  • a binding site comprises any subset of the foregoing residues which are within about 10 angstroms of one another.
  • residues CYS39, ARG41 , SER42, PRO60, GLU62, LYS220, and ASP230 comprise such a binding site.
  • this class comprises compounds containing one or more moieties which are each capable of interacting with one or more of the foregoing residues, preferably with one or more binding sites defined by the foregoing residues.
  • a subset of such compounds are those which contain at least two moieties, of which at least one is a substituted or unsubstituted phosphate or phosphate mimic (e.g., a substituted or unsubstituted phosphonate moiety).
  • the substituent may be alkyl, aryl, or arylalkyl .
  • alkyl is intended to include both saturated and unsaturated straight chain, branched, cyclic, or polycyclic aliphatic hydrocarbons which may contain oxygen, sulfur, or nitrogen in place of one or more carbon atoms, and which are optionally substituted with one or more functional groups selected from the group consisting of hydroxy, C-
  • Alkyl groups are preferably Iower alkyl, i.e. containing 1 to 8 carbon atoms.
  • aryl is intended to include stable cyclic, heterocyclic, polycyclic, and polyheterocyciic unsaturated C3-C-14 moieties, exemplified but not limited to phenyl, biphenyl, naphthyl, pyridyl, furyl, thiophenyl, imidazoyl, pyrimidinyl, and oxazoyl; which may further be substituted with one to five members selected from the group consisting of hydroxy, C-j -C ⁇ alkoxy, C-
  • the ligands may contain one or more amide bonds, but are preferably non-peptidic.
  • the molecular weight of the ligands is under 1200, more preferably under 750, more preferably under 500.
  • Peptides and peptidic molecules comprise two or more naturally occurring ⁇ -amino acids linked by peptide bonds (primary amide bonds, except where the amino acid is proline).
  • the ability of a ligand or a moiety on a iigand to interact with a particular residue or set of residues in a binding site may be determined by noting the proximity of a ligand moiety to a residue of interest.
  • Proximity may be determined by physical methods such as x-ray crystallography or NMR evaluation of a co-complex of the protein and ligand, or may be determined through modeling studies in which the structure of the ligand is docked with the structure of the protein using programs such as described above.
  • a number of commercially available programs are capable of conveniently evaluating a modeled or experimentally determined structure and identifying atoms involved in hydrogen-bonding or hydrophobic interactions.
  • hydrogen bonding (which includes salt bridge and other ionic interactions) occurs across distances of about 2.8 - 3.5 angstroms, more usually up to about 3.2 angstroms, and through donor-H-acceptor angles of about 180° ⁇ 60°. Hydrophobic interactions occur accross distances of up to about 5 angstroms, more preferably up to about 4.5 angstroms and more frequently up to about 4 angstroms, depending on the nature of the atoms involved. Again, any of a number of commercially available computer programs may be used to identify hydrogen bonding and hydrophobic interactions between ligand moieties and protein atoms.
  • Compounds which bind to one or more ZAP famiiy members may be used as biological reagents in binding assays as described herein for functional classification of an SH2-bearing protein, particularly a newly discovered protein, based on ligand specificity.
  • compounds identified as described above can be used to inhibit the occurrence of biological events resulting from molecular interactions mediated by a ZAP famiiy protein containing one or more SH2 domains.
  • This invention thus provides a method and materials for inhibiting (totally or partially) the interaction between such a protein and a natural ligand thereto (i.e., a naturally occurring protein (typically), or a portion or analog thereof, which binds in a cell to the ZAP family protein) or a biological activity mediated by such interaction.
  • a compound identified or obtained as described herein is combined or contacted with the ZAP family protein, such as by introducing the compound into a cell in which the molecular interaction is to be inhibited.
  • the interaction of the ZAP family protein and its natural iigand is inhibited as may be readily detected. Inhibiting such interactions can be useful in research aimed at better understanding the biology of SH2-mediated events.
  • inhibitors of SH2-mediated interactions would be useful, for example, in the diagnosis, prevention or treatment of conditions or diseases resulting from a cellular processes mediated by the interaction of SH2 bearing protein with a natural ligand therefor.
  • a patient can be treated to prevent the occurence or progression of osteoporosis or to reverse its course by administering to the patient in need thereof an SH2 binding or blocking agent which selectively binds Src SH2.
  • SH2 binding or blocking agents may be useful therapeutically, including breast cancer where the SH2 domain-containing proteins Src, PLCg and Grb7 have been implicated.
  • inhibitors of interactions involving ZAP-70 which is believed to be involved in activation of T-cells, would be useful as an immunosuppressant in the treatment and prevention of autoimmune diseases and to prevent rejection of skin and organ transplants.
  • Inhibitors of interactions of SYK with natural ligands would be useful in the treatment and prevention of asthma and untoward allergic reactions.
  • An inhibitor selected or identified in accordance with this invention can be formulated into a pharmaceutical composition containing a pharmaceutically acceptable carrier and/or other excipient(s) using conventional materials and means.
  • a composition can be administered to an animal, either human or non-human, for therapy of a disease or condition resulting from cellular events involving a molecular interaction mediated by a ZAP famiiy protein. Administration of such composition may be by any conventional route (parenteral, oral, inhalation, and the like) using appropriate formulations as are well known in this art.
  • the inhibitor of this invention can be employed in admixture with conventional excipients, ie, pharmaceutically acceptable organic or inorganic carrier substances suitable for parenteral administration.
  • a compound identified as described herein may be used in pharmaceutical compositions and methods for treatment or prevention of various diseases and disorders in a mammal in need thereof.
  • Mammals include rodents such as mice, rats and guinea pigs as well as dogs, cats, horses, cattle, sheep, non-human primates and humans.
  • the preferred method of such treatment or prevention is by administering to a mammal an effective amount of the compound to prevent, alleviate or cure said disease or disorder.
  • effective amounts can be readily determined by evaluating the compounds of this invention in conventional assays well-known in the art, including assays described herein.
  • the invention provides methods of treating, preventing and/or alleviating the symptoms and/or severity of a disease or disorder referred to above by administration to a subject of a in an amount effective therefor.
  • the subject will be an animal, including but not limited to animals such as cows, pigs, chickens, etc., and is preferably a mammal, and most preferably human.
  • Various delivery systems are known and can be used to administer the inhibitor, e.g., encapsulation in liposomes, microparticles, microcapsules, etc.
  • One mode of delivery of interest is via pulmonary administration, as detailed more fully infra.
  • Other methods of introduction include but are not limited to intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural and oral routes.
  • the inhibitor may be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and may be administered together with other biologically active agents. Administration can be systemic or local.
  • preferred routes of administration are oral, nasal or via a bronchial aerosol or nebulizer.
  • compositions comprise a therapeutically (or prophylactically) effective amount of the inhibitor, and a pharmaceut ⁇ ically acceptable carrier or excipient.
  • a carrier includes but is not limited to saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof.
  • the carrier and composition can be sterile.
  • the formulation should suit the mode of administration.
  • the composition can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents.
  • the composition can be a liquid solution, suspension, emulsion, tablet, pill, capsule, sustained release formulation, or powder.
  • the composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides.
  • Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc.
  • the composition is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to human beings.
  • compositions for intravenous administration are solutions in sterile isotonic aqueous buffer.
  • the composition may also include a solubilizing agent and a local anesthetic to ease pain at the side of the injection.
  • the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent.
  • the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline.
  • an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.
  • Administration to an individual of an effective amount of the inhibitor can also be accomplished topically by administering the compound(s) directly to the affected area of the skin of the individual.
  • the inhibitor is administered or applied in a composition including a pharmacologically acceptable topical carrier, such as a gel, an ointment, a lotion, or a cream, which includes, without limitation, such carriers as water, glycerol, alcohol, propylene glycol, fatty alcohols, triglycerides, fatty acid esters, or mineral oils.
  • topical carriers include liquid petroleum, isopropyl palmitate, polyethylene glycol, ethanol (95%), polyoxyethylene monolaurate (5%) in water, or sodium lauryl sulfate (5%) in water.
  • Other materials such as anti-oxidants, humectants, viscosity stabilizers, and similar agents may be added as necessary.
  • the inhibitor may be disposed within devices placed upon, in, or under the skin.
  • Such devices include patches, implants, and injections which release the compound into the skin, by either passive or active release mechanisms.
  • the effective dose of the inhibitor will typically be in the range of about 0.01 to about 50 mg/kgs, preferably about 0.1 to about 10 mg/kg of mammalian body weight, administered in single or multiple doses.
  • the inhibitor may be administered to patients in need of such treatment in a daily dose range of about 1 to about 2000 mg per patient.
  • the amount of the inhibitor which will be effective in the treatment or prevention of a particular disorder or condition will depend on the nature of the disorder or condition, and can be determined by standard clinical techniques. In addition, in vitro or in vivo assays may optionally be employed to help identify optimal dosage ranges. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems.
  • the precise dosage level of the inhibitor, as the active component(s), should be determined as in the case of all pharmaceutical treatments, by the attending physician or other health care provider and will depend upon well known factors, including route of administration, and the age, body weight, sex and general health of the individual; the nature, severity and clinical stage of the disease; and the use (or not) of concomitant therapies.
  • the invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention.
  • a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention.
  • Optionally associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceutical or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration.
  • the inhibitor is administered by pulmonary administration, e.g. via aerosolization.
  • This route of administration may be particularly useful for treatment or prophylaxis of bronchial or pulmonary infection or tumors.
  • Pulmonary administration can be accomplished, for example, using any of various delivery devices known in the art (see e.g., Newman, S.P., 1984, in Aerosols and the Lung , Clarke and Davia (eds.), Butterworths, London, England, pp. 197-224; PCT Publication No. WO
  • nebulizers including but not limited to nebulizers, metered dose inhalers, and powder inhalers.
  • Various delivery devices are commercially available and can be employed, e.g., Ultravent nebulizer (Mallinckrodt, Inc., St.
  • Ultrasonic nebulizers tend to be more efficient than jet nebulizers in producing an aerosol of respirable size from a liquid (Smith and Spino, "Pharmacokinetics of Drugs in Cystic Fibrosis," Consensus Conference, Clinical Outcomes for Evaluation of New CF Therapies, Rockville, Maryland, December 10-1 1 , 1992, Cystic Fibrosis Foundation).
  • a nebulizer may be used to produce aerosol particles, or any of various physiologically acceptable inert gases may be used as an aerosolizing agent.
  • Other components such as physiologically acceptable surfactants (e.g., glycerides), excipients (e.g., lactose), carriers, and diluents may also be included.
  • the ZAP-NC was expressed as a glutathione-S-transferase (GST) fusion protein.
  • GST glutathione-S-transferase
  • the DNA sequence encoding residues 1-259 from human ZAP 70 was cloned into the pGex expression vector (D.B. Smith, K.S. Johnson Gene 67, 31-40 (1988))and transformed into E. coli BL21 or E. Coli B834.
  • the ZAP-NC was produced by the growth and induction of two liters of culture (BL21) in BHI medium.
  • the culture was grown at 25°C to an OD 595nm of 0.8 and induced with 1 mM IPTG for 5 hours.
  • the selenomethionyl (SeMet) ZAP-NC was produced using the auxotrophic strain of E. coli 834 (D.J. Leahy, H.P. Erickson, I. Aukhil, P. Joshi, W. Hendrickson Proteins 19 48-54 (1994)) with the selenomethionine replacing the methionine in a defined media.
  • the SeMet ZAP-NC was grown in 10 liters of defined media (J.O.Boies, W.H. Tolleson, J.C. Schmidt, R.B. Dunlap, J.D. Odom, J. Biol. Chem.
  • the GST fusion proteins were isolated using glutathione agarose and then cleaved with thrombin.
  • ZAP-NC was separated from the GST by binding the tandem SH2 domain to a phosphotyrosine agarose column and eluting with a salt gradient. Subsequently the ZAP-NC was further purified by hydrophobic interaction chromatography on a phenyl sepharose column. The protein was stored under argon with 500mM NaCI and 10mM dithiothreitol at 4°C. A typical purification is listed below. Both the ZAP-NC and the SeMet ZAP-NC were judged to be >95% pure by N- terminal analysis and SDS gel electrophoresis. Mass spectrometric analysis of the ZAP-NC and SeMet ZAP-NC indicated >95% incorporation of the selenomethione.
  • Buffer A Buffer E Buffer G:
  • Buffer B 20mM Tris pH 7.6 20mM Tris pH 8 PBS/0.5% Triton 2M NaCI 5mM DTT

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Abstract

L'invention concerne la ZAP-70 de l'homme et, en particulier, la région de ZAP-70 contenant les domaines tandem d'homologie-2 de Src ('SH2'), ses formes cristallines, à ligand ou sans ligand, qui sont particulièrement utiles pour déterminer la structure tridimensionnelle de la protéine. La structure tridimensionnelle de la région tandem de SH2 de la ZAP fournit des informations utiles à la conception de compositions pharmaceutiques inhibant la fonction biologique de la ZAP et d'autres membres de la famille ZAP de protéines contenant les domaines de SH2, en particulier les fonctions biologiques induites par des interactions moléculaires impliquant un ou deux domaines de SH2.
PCT/US1996/013918 1995-08-30 1996-08-30 Proteines cristallines de la famille zap Ceased WO1997008300A1 (fr)

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JP9510577A JPH11510061A (ja) 1995-08-30 1996-08-30 結晶性ｚａｐファミリータンパク質
AU69606/96A AU6960696A (en) 1995-08-30 1996-08-30 Crystalline zap family proteins
EP96930632A EP0847443A1 (fr) 1995-08-30 1996-08-30 Proteines cristallines de la famille zap

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US297295P 1995-08-30 1995-08-30
US60/002,972 1995-08-30
US331295P 1995-09-06 1995-09-06
US60/003,312 1995-09-06

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WO1997008300A9 WO1997008300A9 (fr) 1997-05-22

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Cited By (7)

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Publication number Priority date Publication date Assignee Title
WO1998020024A1 (fr) * 1996-11-04 1998-05-14 Merck Frosst Canada & Co. Ligands pour dosage par liaison competitive
US5834228A (en) * 1997-02-13 1998-11-10 Merck & Co., Inc. Method for identifying inhibitors for apopain based upon the crystal structure of the apopain: Ac-DEVD-CHO complex
WO1999040182A3 (fr) * 1998-02-04 1999-10-07 Immunex Corp Enzyme de conversion cristalline du facteur tnf alpha et ses utilisations
US6207397B1 (en) 1996-04-18 2001-03-27 Ariad Pharmaceuticals, Inc. In vitro fluorescence polarization assay
US6842704B2 (en) 1998-02-04 2005-01-11 Immunex Corporation Crystalline TNF-α-converting enzyme and uses thereof
US7833525B2 (en) 2000-12-28 2010-11-16 Bhami Shenoy Crystals of whole antibodies and fragments thereof and methods for making and using them
US7998477B2 (en) 2001-06-21 2011-08-16 Althea Technologies Inc. Spherical protein particles and methods for making and using them

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1360286A2 (fr) * 2000-09-19 2003-11-12 Chiron Corporation Caracterisation de la proteine gsk-3 $g(b) et ses procedes d'utilisation
WO2004066921A2 (fr) * 2003-01-21 2004-08-12 Smithkline Beecham Corporation Co-cristal d'erbb4

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* Cited by examiner, † Cited by third party
Title
A VON BONIN ET AL.: "The beta-D sheet of the Lck-derived SH2 domain determine specificity of the interaction with tyrosine-phosphorylated ligands in Ramos B cells", JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 269, no. 52, 20 December 1994 (1994-12-20), MD US, pages a33035 - 33041, XP002023959 *
E DHARM ET AL.: "Large-scale purification and characterization of the 29,000 dalton dual-SH2 domains of the protein tyrosine kinase Syk expressed in E. coli", FASEB JOURNAL FOR EXPERIMENTAL BIOLOGY, vol. 10, no. 3, 8 March 1996 (1996-03-08), BETHESDA, MD US, pages a164, XP002023960 *
G WAKSMAN ET AL.: "Binding of a high-affinity phosphotyrosyl peptide ti the Src SH2 domain; crystal structures of the complexed and peptide-free forms", CELL, vol. 72, 12 March 1993 (1993-03-12), NA US, pages 779 - 790, XP002023957 *
G WAKSMAN ET AL.: "Crystal structure of the phosphotyrosine recognition domain SH2 of v-src complexed with tyrosine-phosphorylated peptides", NATURE, vol. 358, 20 August 1992 (1992-08-20), LONDON GB, pages 646 - 653, XP002023958 *
M H HATADA ET AL.: "Molecular basis for interaction of the protein tyrosine kinase ZAP-70 with the T-cell receptor", NATURE, vol. 377, 7 September 1995 (1995-09-07), LONDON GB, pages 32 - 38, XP002023961 *
S MAIGNAN ET AL.: "Crystal structure of the mammalian Grb2 adaptor", SCIENCE, vol. 268, 14 April 1995 (1995-04-14), LANCASTER, PA US, pages 291 - 293, XP002023956 *

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6207397B1 (en) 1996-04-18 2001-03-27 Ariad Pharmaceuticals, Inc. In vitro fluorescence polarization assay
WO1998020024A1 (fr) * 1996-11-04 1998-05-14 Merck Frosst Canada & Co. Ligands pour dosage par liaison competitive
US5834228A (en) * 1997-02-13 1998-11-10 Merck & Co., Inc. Method for identifying inhibitors for apopain based upon the crystal structure of the apopain: Ac-DEVD-CHO complex
WO1999040182A3 (fr) * 1998-02-04 1999-10-07 Immunex Corp Enzyme de conversion cristalline du facteur tnf alpha et ses utilisations
US6842704B2 (en) 1998-02-04 2005-01-11 Immunex Corporation Crystalline TNF-α-converting enzyme and uses thereof
US7833525B2 (en) 2000-12-28 2010-11-16 Bhami Shenoy Crystals of whole antibodies and fragments thereof and methods for making and using them
US9310379B2 (en) 2000-12-28 2016-04-12 Ajinomoto Althea, Inc. Methods of crystallizing antibodies
US7998477B2 (en) 2001-06-21 2011-08-16 Althea Technologies Inc. Spherical protein particles and methods for making and using them

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EP0847443A1 (fr) 1998-06-17

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