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WO2005045019A1 - Structure tridimensionnelle de la cathepsine e, procedes et utilisation de ladite structure - Google Patents

Structure tridimensionnelle de la cathepsine e, procedes et utilisation de ladite structure Download PDF

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Publication number
WO2005045019A1
WO2005045019A1 PCT/EP2004/012560 EP2004012560W WO2005045019A1 WO 2005045019 A1 WO2005045019 A1 WO 2005045019A1 EP 2004012560 W EP2004012560 W EP 2004012560W WO 2005045019 A1 WO2005045019 A1 WO 2005045019A1
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atom
cathepsin
wat
phe
molecular weight
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Jörg EDER
Nils Ostermann
Susanne Worpenberg
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Novartis Pharma GmbH Austria
Novartis AG
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Novartis Pharma GmbH Austria
Novartis AG
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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
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6478—Aspartic endopeptidases (3.4.23)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2299/00—Coordinates from 3D structures of peptides, e.g. proteins or enzymes

Definitions

  • the present invention relates to the aspartyl protease Cathepsin E (Cat E), in particular, the three-dimensional structure of human Cathepsin E. Further, the invention provides methods of isolation, expression and purification of the recombinant human aspartyl protease Cathepsin E for the use in making crystals comprising Cathepsin E. The invention also relates to the use of the three-dimensional structure of Cathepsin E for identifying and designing ligands or low molecular weight compounds which inhibit the biological activity of Cathepsin E and/or any other aspartyl protease family member.
  • Cathepsin E is an intracellular, non-Iysosomal aspartic proteinase belonging to the A1 pepsin-like family. In vivo, Cathepsin E is synthesized at neutral pH as inactive zymogen. This precursor form is inhibited by binding of its N-terminal pro-sequence in the active site. Cathepsin E, like most aspartic proteases, auto-activates at acidic pH in a stepwise fashion resulting in the mature, active form of the protease at the end of the activation pathway. The mature enzyme appears to be contained within vesicular structures associated with the endoplasmic reticulum, endosomal compartments of selected cells and the trans-Golgi network.
  • Cathepsin E In mature erythrocytes, Cathepsin E is associated with the cytoplasmic face of the plasma membranes. Postulated physiological roles for cathepsin E include the biogenesis of the vasoconstrictor peptide endothelin and the antigen/invariant chain processing. Further, the enzyme might play a role in neurodegeneration associated with brain ischemia and aging. In contrast to all other members of the A1 aspartyl protease family the mature Cathepsin E exists as a disulfide-linked homodimer with a molecular mass of 84 kDa. However, dimer formation is not required for its catalytic activity.
  • Other members of the human aspartyl protease family include: BACE, BACE 2, renin, Cathepsin D, Pepsin A, Pepsin C and Napsin A. While structural information is available for BACE, Renin, Cathepsin D, Pepsin A and Pepsin C, there has been no description of the crystal structure of Cathepsin E in any species described prior to the present invention. The present invention describes for the first time to our knowledge, the three dimensional structure of human Cathepsin E determined by crystallization of Cathepsin E and the use of this structural information for identifying and designing inhibitors for Cathepsin E and other aspartyl protease family members.
  • the present invention provides the three-dimensional structure of Cathepsin E thereby enabling the identification and design of ligands or low molecular weight molecules, particularly inhibitors of aspartyl protease family members.
  • a three-dimensional structure of Cathepsin E comprising the atomic structure coordinates of Table 1 is provided.
  • a crystal of Cathepsin E comprising the sequence of SEQ ID. No. 1 , SEQ ID. No. 2, or a mutant, fragment or a homologue of either SEQ ID No. 1 or SEQ ID No. 2 is provided.
  • at least the ligand binding site is provided.
  • a crystal of cathepsin E bound to at least one ligand or low molecular weight compound is provided.
  • a computer readable medium comprising data storage material encoded with computer readable data wherein said data comprises the atomic coordinates of Table 1 comprising Cathepsin E.
  • the present invention also provides a method for making a crystal of Cathepsin E comprising:
  • Cathepsin E comprising SEQ ID No.1 or SEQ ID No. 2 in a suitable host cell
  • ii purification and, if required, refolding, of Cathepsin E.
  • at least the prosequence is expressed together with the mature Cathepsin E in a suitable host cell.
  • the prosequence or part of the prosequence is removed prior to crystallisation, (iii) exposure of the purified and folded Cathepsin E to conditions suitable for crystalisation
  • At least the prosequence comprising amino acids 19-53 of SEQ ID No. 1 or amino acids 24-58 of SEQ ID No.2 is expressed together with the mature Cathepsin E comprising amino acids 54-396 of SEQ ID No. 1 or amino acids 59-401 of SEQ ID No. 2 in a suitable host cell.
  • the prosequence or part of the prosequence comprising amino acids 19-53 of SEQ ID No. 1 or amino acids 24-58 of SEQ ID No.2 is removed at any step prior to crystallisation.
  • a mutant, fragment or homologue of SEQ ID No.1 or SEQ ID No. 2 may be used for making a crystal of Cathepsin E.
  • At least one ligand or low molecule weight chemical compound may be bound to Cathepsin E at any step prior to crystallisation.
  • the invention provides a method of determining the three-dimensional structure of Cathepsin E comprising:
  • the invention provides a method of determining the three-dimensional structure of a complex comprising Cathepsin E (SEQ ID No. 1 or SEQ ID No.2), a mutant, fragment or homologue thereof bound to at least one ligand comprising: (i) obtaining x-ray diffraction data for crystals of the complex (ii) utilising the atomic coordinates of Table 1 in whole or in part to define the three- dimensional structure of the complex.
  • the invention provides a method of determining the three dimensional structure of a member of the aspartyl protease family comprising: utilizing the atomic coordinates of Table 1 in whole or in part to determining the three dimensional structure of a member of the aspartyl protease family.
  • the invention provides a method of identifying a ligand or low molecular weight compound that binds to Cathepsin E or any other aspartyl protease family member comprising:
  • a method of identifying a ligand or low molecular weight compound that selectively binds to Cathepsin E comprising:
  • a method for identifying a ligand or low molecular weight compound which does not bind to Cathepsin E but selectively binds to another aspartyl protease family member comprising:
  • a ligand or low molecular weight compound which inhibits the biological activity of Cathepsin E or any other aspartyl protease family member is preferred. Most preferred are highly selective inhibitors which inhibit one aspartyl protease family member selectively.
  • a method of designing a ligand or low molecular weight compound capable of binding to Cathepsin E or any other aspartyl protease comprising:
  • a ligand or low molecular weight compound is designed which inhibits the activity of Cathepsin E or any other aspartyl protease family. Most preferred are highly selective inhibitors which inhibit one aspartyl protease family member selectively.
  • the candidate ligands or low molecular weight compounds are screened and designed in silico.
  • a pharmaceutical composition comprising a ligand identified or designed by any of the aforementioned methods is provided for use in preventing or treating of diseases and conditions involving an aspartyl protease family member.
  • the full-length sequence of human Cathepsin E is given by SEQ ID No.1 (see: Genbank Accession number NP 001901 ; Gl:4503145, Swissprot Accession number P14091).
  • Amino acids 1-18 represent the N-terminal signal sequence
  • amino acids 19-53 represent the pro- sequence
  • amino acids 54-396 represent the mature Cathepsin E.
  • a construct of human Cathepsin E is given by SEQ ID No.2.
  • Amino acids 1-23 represent the N-terminal extension
  • amino acids 24-58 represent the pro-sequence
  • amino acids 59-401 represent the mature Cathepsin E
  • amino acids 402-409 represent the C-terminal extension.
  • the amino acids of the pro-sequence and mature Cathepsin E are identical for SEQ ID. 1 and SEQ ID. No. 2, but have different amino acid numbering due to the difference in the length of the N-terminal sequences.
  • SEQ ID. No. 3 and SEQ ID No. 4 represent primers to introduce the C-terminal extension.
  • the C-terminal extension is used to facilitate purification of Cathepsin E.
  • the parameters characterising the unit cell may vary within a limited range, for example, a,b,c each vary by up to 5 Angstroms.
  • the space group of the present invention is P4 1 2 1 2 tetragonal.
  • unit cell refers to the basic shape block.
  • the entire volume of a crystal is constructed by regular assembly of such blocks.
  • Each unit cell comprises a complete representation of the unit of pattern, the repetition of which builds up the crystal.
  • space group refers to the arrangement of symmetry elements of a crystal.
  • ligand refers to a molecule or group of molecules that bind to one or more specific sites of Cathepsin E or another aspartyl protease family member. Preferred ligands are those that selectively bind to the active site of only one aspartyl protease family member. Most preferred are ligands that selectively bind to the active site of Cathepsin E. Ligands according to the invention are preferably low molecular weight molecules.
  • low molecular weight compound refers to preferably organic compounds generally having a molecular weight less than about 1000, more preferably less than about 600. Most preferably, said low molecular weight compounds or ligands selectively inhibit biological activity of Cathepsin E or another aspartyl protease family member.
  • peptide or “peptide derivative” are intended to embrace a “peptidomimetic” or “peptide analogue” which complement the three- dimensional structure of the binding site of Cathepsin E or can be designed with improved physical or chemical properties to bind with the three-dimensional binding site of the Cathepsin E as provided in the present invention.
  • mutant refers to differences within the wild-type sequence of Cathepsin E set forth in SEQ. ID No. 1 by deletion, insertion, extension, or replacement of one or more selected amino acids.
  • the term "mutant” also refers to a polypeptide, whose amino acid sequence differs from the sequence given in SEQ ID No.2 by deletion, insertion or preferably replacement of one or more selected amino acids.
  • a Cathepsin E mutant of the present invention is preferably at least 50% homologous to SEQ ID No. 2, more preferably at least 80% homologous to SEQ ID No. 2 most preferably at least 90% homologous to SEQ ID No. 2.
  • a “fragment” of Cathepsin E according to the invention comprises more than 50% of the full- length sequence of Cathepsin E according to SEQ ID No.1 or SEQ ID No. 2, more preferably at least 80% of the full length sequence of Cathepsin E according to SEQ ID No.1 or SEQ ID No. 2, most preferably at least 90% of the full-length sequence of Cathepsin E according to SEQ ID No. 1 or SEQ ID No. 2.
  • a "N-terminal extension" of Cathepsin E according to the invention comprises the addition of amino acids at the N terminus of the full length Cathepsin E.
  • the N terminal extension of SEQ ID No.2 is represent by amino acids 1-23.
  • a "C-terminal extension" of Cathepsin E according to the invention comprises the addition of 8-10 amino acids at the C-terminus of the full-length Cathepsin E.
  • the C- terminal extension of SEQ ID No. 2 is represented by amino acids 402-409.
  • Cathepsin E may be crystallisable with or without at least one ligand. It may also be crystallisable with or without the pro-sequence or parts of the pro-sequence of Cathepsin E. According to the present invention, Cathepsin E crystals are stable if kept under suitable conditions. For example, the crystals are stable in there mother liquor at 20°C for at least 3-4 weeks. Preferable storage is frozen in liquid nitrogen.
  • Cathepsin E a mutant, fragment or homologue thereof is advantageously obtained by expressing proCathepsin E in a recombinant host cell culture and subsequent refolding and auto-proteolytic cleavage of the pro-sequence.
  • Cathepsin E may be prepared by isolation from natural sources, e.g. cultured human cells or preferably by recombinant heterologous expression. Expression of recombinant Cathepsin E is achievable in eukaryotic or prokaryotic systems.
  • recombinant human Cathepsin E may be expressed in bacteria.
  • the protease may be expressed as a Strep-tag fusion protein, a glutathione-S-transferase (GST) fusion protein, a histidine-tagged fusion protein or as an untagged protein. If desired, the fusion partner is removed before crystallization.
  • the heterologously produced Cathepsin E to be used for crystallization is potentially biologically active. Such ability may be determined by morphological, biochemical or viability analysis well-known in the art.
  • Cathepsin E mutants may be prepared by expression of Cathepsin E DNA previously modified in its coding region by oligo-nucleotide directed mutagenesis.
  • purified Cathepsin E is preferably at least 90 % homogeneous. Protein homogeneity is determinable according to analytical methods well-known in the art, e.g. sequence analysis, electrophoresis, spectroscopic or chromatographic techniques. The purified protein is potentially proteolytically active. Appropriate assays for determining Cathepsin E activity towards a suitable substrate, e.g.
  • Cathepsin E may be complexed with a low molecular weight compound or ligand which is capable of suitably binding to Cathepsin E.
  • a compound inhibiting Cathepsin E activity is determinable employing assays known in the art. Suitable inhibitors include protease inhibitors which act on the catalytic site to inhibit Cathepsin E activity.
  • cystallization can be used in the claimed invention including vapor diffusion, dialysis or batch crystallization.
  • vapor diffusion crystallization a small volume (i.e., a few microliters) of protein solution is mixed with a solution containing a precipitant. This mixed volume is suspended over a well containing a small amount, i.e. about 0.15-1 ml, of precipitant. Vapor diffusion between the drop and the well will result in crystal formation in the drop.
  • the dialysis method of crystallization utilizes a semipermeable size-exclusion membrane that retains the protein but allows small molecules (i.e. buffers and precipitants) to diffuse in and out.
  • small molecules i.e. buffers and precipitants
  • the precipitant is allowed to slowly diffuse through the membrane and reduce the solubility of the protein while keeping the protein concentration fixed.
  • the batch method generally involves the slow addition of a precipitant to an aqueous solution of protein until the solution just becomes turbid, at this point the container can be sealed and left undisturbed for a period of time until crystallization occurs.
  • the precipitant and the target molecule solution are simply mixed. Supersaturation is achieved directly rather than by diffusion.
  • the batch technique is performed under oil. The oil prevents evaporation and extremely small drops can be used. For this, the term "microbatch" is used.
  • a modification of this technique is not to use paraffin oil (which prevents evaporation completely) but rather use silicone oil or a mixture of silicone and paraffin oils so that a slow evaporation is possible.
  • the claimed invention can encompass any and all methods of crystallization.
  • One skilled in the art can choose any of such methods and vary the parameters such that the chosen method results in the desired crystals.
  • One preferred method of crystallization of Cathepsin E involves mixing a Cathepsin E solution with a "reservoir buffer", with a lower concentration of the precipitating agent necessary for crystal formation.
  • concentration of the precipitating agent has to be increased, e.g. by addition of precipitating agent, for example by titration, or by allowing the concentration of precipitating agent to balance by diffusion between the crystallization buffer and a reservoir buffer.
  • Diffusion may be achieved e.g.
  • vapour diffusion techniques allowing diffusion of water in the common gas phase.
  • Known techniques are e.g. vapour diffusion methods, such as the "hanging drop” or the “sitting drop” method.
  • vapour diffusion method a drop of crystallization buffer containing the protein is hanging above or sitting beside a much larger pool of reservoir buffer.
  • the balancing of the precipitating agent can be achieved through a semipermeable membrane (dialysis method) that separates the crystallization buffer from the reservoir buffer and prevents dilution of the protein into the reservoir buffer.
  • Formation of Cathepsin E crystals can be achieved under various conditions which are essentially determined by the following parameters: pH, presence of salts and additives, precipitating agent, protein concentration and temperature.
  • the pH may range, for example, from about 3.0 to 11.0.
  • the present invention also relates to a computer readable medium having stored a model of the Cathepsin E crystal structure.
  • said model is built from all or part of the X-ray diffraction data.
  • the atomic coordinates are shown in Table 1.
  • the present invention provides the structure coordinates of human Cathepsin E.
  • structure coordinates or "atomic coordinates” refers to mathematical coordinates derived from the mathematical equations (Fourier transformation) related to the diffraction pattern obtained on a monochromatic beam of X-rays by the atoms (scattering centers) of a crystal comprising a Cathepsin E.
  • the diffraction data are used to calculate an electron density map of the repeating unit of the crystal.
  • the electron density maps are used to establish the positions of the individual atoms within the unit cell of the crystal.
  • 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, CD, DVD 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, CD, DVD etc.
  • data defining the three dimensional structure of a protein of Cathepsin E, 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.
  • a three-dimensional Cathepsin E model is obtainable from a Cathepsin E crystal comprising Cathepsin E, mutant, fragment or homologue thereof.
  • Such a model can be built or refined from all or part of the Cathepsin E structure data of the present invention using the x-ray diffraction coordinates, particularly the atomic structure coordinates of Table 1.
  • the knowledge obtained from the three-dimensional model of Cathepsin E can be used in various ways. For example, it can be used to identify chemical entities, for example, small organic and bioorganic molecules such as peptidomimetics and synthetic organic molecules that bind to Cathepsin E and preferably block or prevent a Cathepsin E mediated or associated process or event, or that bind to another aspartyl protease or prevent another aspartyl protease mediated or associated process or event.
  • the skilled artisan constructs a model of the Cathepsin E. For example, every atom can be depicted as a sphere of the appropriate van der Waals radius, and a detailed surface map of Cathepsin E can be constructed.
  • Chemical entities that have a surface that mimics the accessible surface of the catalytic binding site of Cathepsin E can be constructed by those skilled in the art.
  • the skilled artisan can screen three-dimensional structural databases of compounds to identify those compounds that position appropriate functional groups in similar three dimensional structural arrangement, then build combinatorial chemistry libraries around such chemical entities to identify those with high affinity to the catalytic binding site of Cathepsin E.
  • Ligands or small molecular compounds can be identified from screening compound databases or libraries and using a computational means to form a fitting operation to a binding site on Cathepsin E.
  • the three dimensional structure of Cathepsin E as provided in the present invention in whole or in part by the structural coordinates of Table 1 can be used together with various docking programs.
  • the potential inhibitory or binding effect of a chemical entity on Cathepsin E may be analyzed prior to its actual synthesis and testing by the use of computer-modeling techniques. If the theoretical structure of the given chemical entity suggests insufficient interaction and association between it and Cathepsin E, the need for synthesis and testing of the chemical entity is obviated. However, if computer modeling indicates a strong interaction, the molecule may then be synthesized and tested for its ability to bind to Cathepsin E. Thus, expensive and time-consuming synthesis of inoperative compounds may be avoided.
  • An inhibitory or other binding compound of Cathepsin E may be computationally evaluated and designed by means of a series of steps in which chemical entities or fragments are screened and selected for their ability to associate with the individual binding sites of Cathepsin E.
  • chemical entities or fragments are screened and selected for their ability to associate with the individual binding sites of Cathepsin E.
  • one skilled in the art may use one of several methods to screen chemical entities or fragments for their ability to associate with Cathepsin E. This process may begin by visual inspection of, for example, the binding site on a computer screen based on the structural coordinates of Table 1 in whole or in part. Selected fragments or chemical entities may then be positioned in a variety of orientations, or "docked,” within the catalytic binding site of Cathepsin E.
  • Docking may be accomplished using software such as Quanta and SYBYL, followed by energy minimization and molecular dynamics with standard molecular mechanics force fields, such as CHARMM or AMBER.
  • Specialized computer programs may be of use for selecting interesting fragments or chemical entities. These programs include, for example, GRID, available from Oxford University, Oxford, UK; 5 MCSS or CATALYST, available from Molecular Simulations, Burlington, MA; AUTODOCK, available from Scripps Research Institute, La Jolla, CA; DOCK, available from University of California, San Francisco, CA, and XSITE, available from University College of London, UK.
  • the structure of a crystalline Cathepsin E or portion thereof can for example, be bound to one or more ligands or low molecular weight compounds to form a complex.
  • molecular replacement refers to a method that involves generating a preliminary structural model of a crystal whose structural coordinates are unknown, by orienting and positioning a molecule whose structural coordinates are known, e.g., the Cathepsin E coordinates within the unit cell of the unknown crystal, so as to best account for the observed diffraction pattern of the unknown crystal. Phases can then be calculated from this model, and combined with the observed amplitudes to give an approximated Fourier synthesis of the structure whose coordinates are unknown. This in turn can be subject to any of the several forms of refinement to provide a final accurate structure.
  • molecular replacement may be used to determine the structural coordinates of a crystalline co complex, unknown ligand, mutant, or homolog, or of a different crystalline form of Cathepsin E. Additionally, the claimed crystal and its coordinates may be used to determine the structural coordinates of a chemical entity that associates with Cathepsin E.
  • Homology modeling involves constructing a model of an unknown structure using structural coordinates of one or more related proteins, protein domains and/or one subdomain. 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 by replacement of amino acids or other components by those of the related structure to be solved.
  • Molecular replacement uses a molecule having a known structure.
  • the three-dimensional structure of Cathepsin E provided in whole or in part in Table 1 in a machine-readable form on a data-carrier can be used 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. Once positioned, 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 approximate structure can be fine-tuned to yield a more accurate and often higher resolution structure using various refinement techniques.
  • 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.
  • the present invention also enables homologues and mutants of Cathepsin E and the solving of their crystal structure.
  • the effects of site-specific mutations can be predicted. More specifically, the structural information provided herein permits the identification of desirable sites for amino acid modification, particularly amino acid mutation resulting in substitutional, insertional or deletional variants.
  • Such variants may be designed to have special properties, particularly properties distinct from wild-type Cathepsin E, such as altered catalytic activity. Substitutions, deletions and insertions may be combined to arrive at a desired variant.
  • Such variants can be prepared by methods well-known in the art, e.g. starting from wild-type Cathepsin E or by de novo synthesis.
  • Cathepsin E may also crystallize in a form different from the one disclosed herein.
  • the structural information provided, for example, in SEQ ID No. 2 and Table 1 in whole or in part, is also useful for solving the structure of other crystal forms. Furthermore, it may serve to solve the structure of a Cathepsin E mutant, a Cathepsin E co-complex or a sufficiently homologous protein such as another aspartyl protease family member.
  • the Cathepsin E structural information provided herein is useful for the design of ligands or small molecule compounds which are capable of selectively interacting with Cathepsin E and thereby specifically modulating the biological activity of Cathepsin E. Furthermore, this information can be used to design and prepare Cathepsin E mutants, e.g. mutants with altered catalytic activity, model the three-dimensional structure and solve the crystal structure of proteins, such as Cathepsin E homologues, Cathepsin E mutants or Cathepsin E co-complexes, involving e.g. molecular replacement.
  • the present invention also relates to the chemical entity or ligand identified by such method.
  • the present invention may also be used to design ligands or low molecular weight compounds which bind to another aspartyl protease family member using the atomic coordinates of Table 1 in whole or in part to determine the three-dimensional structure of a aspartyl protease family member.
  • the present invention may also be used to design ligands or low molecular weight compounds which specifically inhibit one aspartyl protease family member and which specifically do not bind to Cathepsin E.
  • One approach enabled by this invention is the use of the structural coordinates of Cathepsin E to design chemical entities that bind to or associate with Cathepsin E and alter the physical properties of the chemical entities in different ways.
  • properties such as, for example, solubility, affinity, specificity, potency, on/off rates, or other binding characteristics may all be altered and/or maximized.
  • One may design desired chemical entities by probing an Cathepsin E crystal comprising Cathepsin E with a library of different entities to determine optimal sites for interaction between candidate chemical entities and Cathepsin E. For example, high-resolution x-ray diffraction data collected from crystals saturated with solutes allows the determination of where each type of solute molecule adheres.
  • Small molecules that bind tightly to those sites can then be designed and synthesized and tested for the desired activity. Once the desired activity is obtained, the molecule can be further altered to maximize desired properties.
  • the invention also contemplates computational screening of small-molecule databases or designing of chemical entities that can bind in whole or in part to Cathepsin E. They may also be used to solve the crystal structure of mutants, co-complexes, or the crystalline form of any other molecule homologous to, or capable of associating with, at least a portion of Cathepsin E.
  • One method that may be employed for this purpose is molecular replacement.
  • An unknown crystal structure which may be any unknown structure, such as, for example, another crystal form of Cathepsin E, an Cathepsin E mutant or peptide, or a co-complex with Cathepsin E, or any other unknown crystal of a chemical entity that associates with Cathepsin E that is of interest, may be determined using the whole of part of the structural coordinates set forth in Table 1. This method provides an accurate structural form for the unknown crystal far more quickly and efficiently than attempting to determine such information without the invention herein.
  • candidate ligands are screened in silico.
  • the information obtained can be used to obtain specific inhibitors of Cathepsin E or any other aspartyl protease family member.
  • a method is provided to select inhibitors specific for only one aspartyl protease family member. For example, an inhibitor which inhibits only Cathepsin E but not other aspartyl protease family members.
  • a method is provided to design ligands which inhibit the activity of Cathepsin E or any other aspartyl protease family member.
  • a method is provided to design a selective inhibitor which is specific for only one aspartyl protease family member, for example, specific for only Cathepsin E.
  • the design of chemical entities that inhibit Cathepsin E generally involves consideration of at least two factors.
  • the chemical entity must be capable of physically or structurally associating with Cathepsin E, preferably at the catalytic site of Cathepsin E.
  • the association may be any physical, structural, or chemical association, such as, for example, covalent or non-covalent binding, or van der Waals, hydrophobic, or electrostatic interactions.
  • the chemical entity must be able to assume a conformation that allows it to associate with Cathepsin E, preferentially at the catalytic site of Cathepsin E. Although not all portions of the chemical entity will necessarily participate in the association with Cathepsin E, those non-participating portions may still influence the overall conformation of the molecule. This in turn may have a significant impact on the desirability of the chemical entity.
  • conformational requirements include the overall three-dimensional structure and orientation of the chemical entity in relation to all or a portion of the binding site.
  • the efficiency with which that compound may bind to Cathepsin E may be tested and modified for the maximum desired characteristic(s) using computational or experimental evaluation.
  • Various parameters can be maximized depending on the desired result. These include, but are not limited to, specificity, affinity, on/off rates, hydrophobicity, solubility, and other characteristics readily identifiable by the skilled artisan.
  • the present invention also relates to identification of compounds which inhibit Cathepsin E or any other aspartyl protease family members activity.
  • said compounds are useful in preventing or treating disorders mediated by Cathepsin E or other aspartyl protease family members, for example, acute or chronic rejection or organ or tissue allografts or xenografts, atherosclerosis, vascular occlusion due to vascular injury such as angioplasty, restenosis, hypertension, heart failure, chronic obstructive pulmonary disease, CNS disease such as Alzheimer disease or amyotrophic lateral sclerosis, cancer, infectious diseases such as AIDS, septic shock or adult respiratory distress syndrome, ischemia/reperfusion injury e.g.
  • T-cell mediated acute or chronic inflammatory diseases or disorders or autoimmune diseases for example, rheumatoid arthritis, osteoarthritis, systemic lupus erythematosus, Hashimoto's thyroidis, multiple sclerosis, myasthenia gravis, diabetes type I or II and disorders associated therewith, respiratory diseases such as asthma or inflammatory lung injury, inflammatory liver injury, inflammatory glomerular injury, cutaneous manifestations of immunologically- mediated disorders or illnesses, inflammatory and hyperproliferative skin diseases (such as psoriasis, atopic dermatitis, allergic contact dermatitis, irritant contact dermatitis, and further eczematous dermatitis, seberrhoeic dermatitis), inflammatory eye diseases, e.g.
  • the required dosage will depend on the mode of administration, the particular condition to be treated and the desired effect. In general, satisfactory results are to be obtained systematically at daily dosages from about 0.1 to about 100 mg/kg body weight.
  • a daily dosage in a larger mammal e.g. human
  • the compounds may be administered by any conventional route, in particular enterally e.g. orally, e.g.
  • compositions comprising said compound in free form or in a pharmaceutically acceptable salt form in association with at least one pharmaceutically acceptable carrier or diluent may be manufactured in conventional manner by mixing with a pharmaceutically acceptable carrier or diluent.
  • Unit dosage forms for oral administration contain, for example, from about 0.1 mg to about 500 mg of active substance.
  • Topical administration is e.g. to the skin.
  • a further form of topical administration is to the eye.
  • Compounds may be administered in free form or in a pharmaceutically acceptable salt form. Such salts may be prepared in a conventional manner and exhibit the same order of activity as the free compounds.
  • the present invention enables the use of molecular design techniques, particularly the rational drug design approach, to prepare new or improved chemical entities and compounds, including Cathepsin E inhibitors, capable of irreversibly or reversibly, modulating Cathepsin E activity.
  • Improved entities or compounds means that these entities or compounds are superior to the "original" or parent compound they are derived from with regard to a property relevant to therapeutic use including suitability for in vivo administration, e.g. cellular uptake, solubility, stability against (enzymatic) degradation, binding affinity or specificity, and the like.
  • Cathepsin E inhibitors which covalently, or preferably non-covalently, bind to Cathepsin E.
  • Such inhibitors may act in a competitive or uncompetitive manner, bind at or close to the active site of Cathepsin E or act allosterically.
  • Cathepsin E modulators the following aspects should be considered: (i) if the candidate compound is capable of physically and structurally associating with Cathepsin E, and/or (ii) if the compound is capable of assuming a conformation allowing it to associate with Cathepsin E.
  • computer modelling techniques are used in the process of assessing these abilities for the modulator as a whole, or a fragment thereof - in order to minimize efforts in the synthesis or testing of unsuccessful candidate compounds. Specialized computer software is well-known in the art.
  • Another design approach is to probe a Cathepsin E crystal with a variety of different chemical entities to determine optimal sites for interaction between candidate Cathepsin E inhibitors and the target enzyme.
  • Yet another possibility which arises from the present invention is to screen computationally small molecule data bases for chemical entities or compounds that are capable of binding, in whole or in part, to Cathepsin E catalytic domain. The quality of fit to the binding site may be judged e.g. by shape complementarity or by estimated interaction energy. Knowledge of the three-dimensional arrangement of the modifications can be then utilized for the design of new Cathepsin E ligands or low molecular weight compounds such as selective inhibitors.
  • Chemical entities that are capable of associating with the aspartyl protease family member may inhibit its interaction with naturally occurring ligands of the protein and may inhibit biological functions mediated by such interaction.
  • biological functions include the biogenesis of the vasoconstrictor peptide endothelin and the antigen/invariant chain processing.
  • Such chemical entities are potential drug candidates.
  • Compounds of the structures selected or designed by any of the foregoing means may be tested for their ability to bind to an aspartyl protease family member, particularly to Cathepsin E, to inhibit the binding to a natural or non-natural ligand therefore, and/or inhibit a biological function mediated by the aspartyl protease family member.
  • the gene of human pro-cathepsin E is inserted into the expression vector pET22b. Insertion of the pro-Cathepsin E gene into pET22b results in 23 additional amino acids at the N- terminus of pro-Cathepsin E (SEQ ID No. 2).
  • the pro-Cathepsin E gene is C-terminally extended to code for 8 additional amino acid residues (WSHPQFEK) in order to facilitate purification by using the Sfrep-tag ® system.
  • the sequence of the primers to introduce the C- terminal extension is given by SEQ ID No. 3 and SEQ ID No. 4.
  • Example 2 Expression of Cathepsin E in E. coli, refolding and purification
  • E. coli cells (BL21(DE3)pLysS harboring the cathepsin E expression plasmid are induced with IPTG. Cells are resuspended in 50 mM Tris/HCl buffer at pH 8.0 and ruptured by sonication. After centrifugation of the homogenate at 16500g for 15 min, the inclusion body- containing pellet is washed twice with the same buffer and then dissolved in 50 mM Tris/HCl buffer at pH 8.0, containing 8 M guanidinium chloride and 100 mM DTE.
  • Pro-cathepsin E is refolded by diluting the clear supernatant at a ratio of 1:150 into 50 mM Tris/HCl buffer at pH 9.5, containing 1 mM EDTA, 0.7 M arginine, 0.5 mM oxidized glutathione and 2 mM reduced glutathione, followed by dialysis against 25 mM Tris/HCl buffer, pH 8.0, 1 mM EDTA.
  • the protein solution is concentrated 10-fold and the pH adjusted to 3.5 with a 1 M sodium citrate solution, pH 3.1.
  • the fractions containing active cathepsin E are pooled and loaded onto a Superdex 200 16/60 gel filtration column which is equilibrated and run with buffer A. Pure cathepsin E is then concentrated to 3 mg/ml and subjected to crystallization trials.
  • Example 3 Crystallization, data collection and structure determination of human Cathepsin E Crystallization Human Cathepsin E is crystallized at 20° by the vapor diffusion method in sitting drops using a cartesian robotics and 96 well plates. 200 nl of the protein solution (3 mg/ml Cathepsin E, 100 mM Tris/HCl at pH 8.0, 150 mM NaCI and 1 mM EDTA) are mixed with 200 nl reservoir solution and equilibrated against 150 ⁇ l of the respective reservoir solution.
  • the protein solution 3 mg/ml Cathepsin E, 100 mM Tris/HCl at pH 8.0, 150 mM NaCI and 1 mM EDTA
  • Diffracting quality crystals can be obtained after approximately five month in the following two reservoir solutions: a) 16% PEG4000, 100 mM Tris/HCl at pH 8.5 and 200 mM MgCI 2 b) 30%) PEG4000, 100 mM sodium citrate at pH 5.6 and 200 mM ammonium sulfate For both crystallization conditions the crystals have a tetragonal bipyramidal morphology.
  • a crystal is cryoprotected by addition of 1 ⁇ l reservoir solution and 0.2 ⁇ l Glycerol to the nano drop and flash-frozen in liquid nitrogen.
  • X-ray diffraction data were collected on a Nonius FR591 rotating anode operating at 5 kW (55 kV and 90 mA) with a MAR345 imaging plate detector at 100 K. 38 images are collected with 1.0° oscillation each, using an exposure time of 60 min per frame and a crystal-to-detector distance of 200 mm.
  • the raw diffraction data are processed with MOSFLM and scaled with SCALA from the CCP4 suite (Collaborative Computational Project, Number 4, 1994).
  • the data collection statistics are summarized in Table 2.
  • Cathepsin E can be solved by the molecular replacement method using the program MOLREP (Collaborative Computational Project, Number 4, 1994) and the coordinates of human Pepsin A (Protein Data Bank accession code 1PSN, Fujinaga et al., 1995) as a search model.
  • MOLREP Collaborative Computational Project, Number 4, 1994
  • Pepsin A Protein Data Bank accession code 1PSN, Fujinaga et al., 1995
  • Using a high resolution data cut off of 3.5 A an unambiguous solution is found in space group P4i2i2 with one cathepsin E molecule in the asymmetric unit (correlation coefficient of 0.44, R-factor of 0.48).
  • the water molecules of the final model are placed using the water-pick protocol as implemented in CNX version 2000.
  • the stereochemical quality of the model is analyzed with the programs CNX version 2000 and PROCHECK (Laskowski et al. 1993).
  • the refinement statistics are summarized in Table 3.
  • Cathepsin E (amino acid numbering corresponding to SEQ ID No.1) comprises the general fold of A1 aspartyl proteases.
  • Cathepsin E contains three topologically distinct reagents, a N-terminal domain (residues Lys67-Val210), a C-terminal domain (residues Thr251-Gln377) and a six-stranded anti-parallel ⁇ -sheet interdomain, connecting the pseudo-twofold-related N- and C-terminal domains. Both, the N- and C- terminal domains contribute an aspartic acid, Asp96 and Asp281 respectively, to the active site.
  • the interdomain consists of amino acids Gly19-Arg27 from the pro-sequence, building the outermost strand of the ⁇ -sheet and being non covalently associated to Cathepsin E, amino acids Asp211-Val250, linking the N- and the C-terminal domains, and the C-terminal amino acids Phe378-Val395.
  • amino acids Arg28-Met53 of the pro-sequence are completely disordered and therefore not visible in the x-ray structure. Additional disordered regions, that have not been considered in the Cathepsin E structure, comprise the N-terminal amino acids Ile54-Ala66, amino acids Glu224-Gly225 as well as amino acids Phe344-Met348.
  • the Cathepsin E structure represents an intermediate 2 state on the activation pathway of aspartyl proteases, similar to that described for Pepsin C with the N-terminal amino acids of mature Cathepsin E (residues Lys67-Glu77) residing in the active site, more precisely, in the unprimed binding site of the active site.
  • the Tyr73 side chain occupies the S1 binding site and makes a hydrogen bond to the carboxylate of Asp96.
  • Leu74 and the backbone carbonyl group of Tyr73 bind to the S3 pocket, while the side chain of Asn72 points towards the S4 pocket.
  • the ⁇ - hairpin structure termed the flap (residues Phe135-Gly146), is displace by approximately 4 A in comparison to the position, observed in known aspartyl protease structures complexed with pepide-based transition state inhibitors.
  • the loop Val169-Ala179 shielding the combined S1-S3 hydrophobic binding pocked from the solvent, is also displaced to some extend.
  • the primed binding site of the active site is not occupied and is observed in an active conformation with the Proline rich loop, flanking the S2' binding pocket (residues Gly357- Leu367), in the closed conformation.
  • ATOM 22 CA ALA P 17 24, .386 -13, .725 17, .695 1. ,00 51, .14 P c
  • ATOM 24 c ALA P 17 24, .023 -12, ,825 16, .523 1, ,00 52, .50 P c
  • ATOM 110 CE LYS A 67 -7, .749 15. .401 44, .729 1, .00 48, .51 A C
  • ATOM 140 N ILE A 71 -7. ,971 8, ,389 35. .126 1, ,00 28, .30 A N
  • ATOM 236 CA ILE A 82 4, .832 -8, .413 32, .408 1, .00 20, .03 A C
  • ATOM 269 CA PRO A 87 8, .456 -16, .737 41, .258 1, .00 21, .84 A C
  • ATOM 272 C PRO A 87 7, .714 -15, .547 40 .637 1. .00 21, .60 A c
  • ATOM 276 CA PRO A 88 6, .915 -14, .422 38, .601 1. .00 20, .92 A c
  • ATOM 282 CA GLN A 89 3, .827 -12, .393 39, .475 1, .00 19, .51 A C
  • ATOM 294 OD1 ASN A 90 3. .359 -13. ,364 34. ,679 1. ,00 22. ,89 A 0
  • ATOM 412 CA PRO A 105 -0.963 -2.202 42, .677 1.00 17, .56 A C
  • ATOM 468 N ALA A 113 • 11. .260 -0, .688 44, .879 1, .00 26. ,75 A N
  • ATOM 469 CA ALA A 113 • 10. .808 -0, .613 43, .496 1, .00 27. ,06 A C
  • ATOM 472 O ALA A 113 -9. ,366 -1. ,854 42. ,022 1. ,00 28. 34 A O
  • ATOM 534 CA GLN A 121 3.124 -9, .368 47, .019 1, .00 24 .57 A C
  • ATOM 610 N PRO A 131 14. 430 -0. 105 48.255 1. 00 31. 42 A N
  • ATOM 654 CA ILE A 137 2.965 10.224 40. ,748 1.00 26. ,53 A C
  • ATOM 718 CA GLY A 146 3, .091 4.186 47 .564 1.00 17, .25 A C
  • ATOM 721 N ILE A 147 5, .206 3.027 47 .850 1.00 17, .71 A N
  • ATOM 730 CA ILE A 148 9, .395 2.061 45, .677 1.00 16, .15 A C
  • ATOM 742 CA ALA A 150 13. ,214 -3.349 43. ,363 1.00 16. ,67 A C
  • ATOM 746 N ASP A 151 13. .160 -5.361 42. ,026 1.00 19. ,29 A N
  • ATOM 750 OD1 ASP A 151 12, .261 -9, .569 41. .928 1, .00 24 .05 A 0
  • ATOM 758 CD GLN A 152 13, .578 -12, .504 38, .770 1, .00 26 .04 A c
  • ATOM 805 CA THR A 159 14. .528 -9, ,803 32, .211 1, .00 20, .49 A c
  • ATOM 812 CA VAL A 160 15. .785 -6. .642 33. .888 1, .00 20, .65 A c
  • ATOM 826 CA GLY A 162 17. ,168 -4. ,690 40. .068 1. ,00 19. .50 A C
  • ATOM 830 CA GLN A 163 14. ,474 -2. ,716 38. .260 1. ,00 18. ,33 A C
  • ATOM 833 CD GLN A 163 13, .025 -1. ,079 35. .946 1. .00 17, .25 A C
  • ATOM 838 N GLN A 164 13, .581 -0, .854 39, .543 1, .00 18, .66 A N
  • ATOM 839 CA GLN A 164 12. .794 -0, .232 40, .600 1, .00 18, .49 A C
  • ATOM 842 CD GLN A 164 15, .393 1, .913 42, .568 1, .00 22, .09 A C
  • ATOM 848 CA PHE A 165 9, .066 0, .365 40, .396 1, .00 16, .81 A C
  • ATOM 902 CA PRO A 172 -6.567 4.696 42, .602 1, .00 22, .10 A C
  • ATOM 908 CA GLY A 173 -10.078 5.885 41, ,822 1, .00 25, ,95 A C
  • ATOM 912 CA GLN A 174 -13.463 4.156 41. ,906 1. ,00 30. ,80 A C

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Abstract

La présente invention concerne la cathepsine E, en particulier la structure tridimensionnelle de la cathepsine E. Cette invention concerne également des procédés d'expression, de purification et de cristallisation de la cathepsine E en vue de la cristallographie de cette dernière. Ladite invention se rapporte en outre aux coordonnées atomiques d'un complexe de cathepsine E avec des parties de la proséquence.
PCT/EP2004/012560 2003-11-07 2004-11-05 Structure tridimensionnelle de la cathepsine e, procedes et utilisation de ladite structure Ceased WO2005045019A1 (fr)

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Cited By (1)

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CN103757014A (zh) * 2014-01-08 2014-04-30 上海大学 富集Ac/Ds侧翼序列用特异性引物及其富集方法

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BALDWIN ERIC T ET AL: "Crystal structures of native and inhibited forms of human cathepsin D: Implications for lysosomal targeting and drug design", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF USA, NATIONAL ACADEMY OF SCIENCE. WASHINGTON, US, vol. 90, no. 14, 1993, pages 6796 - 6800, XP002147298, ISSN: 0027-8424 *
DATABASE PDB [online] 6 April 2002 (2002-04-06), M.V. RAGHU RAM: "A theoretical Model of human cathepsin E Precursor", XP002322874, retrieved from HTTP://OCA.EBI.AC.UK/OCA-BIN/OCASHORT?ID=1LCG Database accession no. 1LCG *
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103757014A (zh) * 2014-01-08 2014-04-30 上海大学 富集Ac/Ds侧翼序列用特异性引物及其富集方法
CN103757014B (zh) * 2014-01-08 2016-05-25 上海大学 富集Ac/Ds侧翼序列用特异性引物及其富集方法

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