WO2003000726A1 - Structure cristalline de la sous-unite apc10/doc1 du complexe de promotion de l'anaphase chez l'homme - Google Patents

Structure cristalline de la sous-unite apc10/doc1 du complexe de promotion de l'anaphase chez l'homme Download PDF

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WO2003000726A1
WO2003000726A1 PCT/EP2002/006909 EP0206909W WO03000726A1 WO 2003000726 A1 WO2003000726 A1 WO 2003000726A1 EP 0206909 W EP0206909 W EP 0206909W WO 03000726 A1 WO03000726 A1 WO 03000726A1
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apc10
apc
protein
inhibitor
human
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Kerstin Wendt
Uwe Jacob
Robert Huber
Peter Sondermann
Michael Gmachl
Jan-Michael Peters
Christian Gieffers
Hartmut Vodermaier
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Max Planck Gesellschaft zur Foerderung der Wissenschaften eV
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Max Planck Gesellschaft zur Foerderung der Wissenschaften eV
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2299/00—Coordinates from 3D structures of peptides, e.g. proteins or enzymes
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A90/00—Technologies having an indirect contribution to adaptation to climate change
    • Y02A90/10—Information and communication technologies [ICT] supporting adaptation to climate change, e.g. for weather forecasting or climate simulation

Definitions

  • the invention relates to the crystal structure of APC10/DOC1 , a subunit of human anaphase-promoting complex (APC), the use of a crystalline APC10/COC1 subunit in order to obtain crystal structure data enabeling rational drug design and screening for inhibitors of APC, pharmaceutical compositions containing such inhibitors as well as the use of inhibitors to prevent the induction of anaphase.
  • APC human anaphase-promoting complex
  • the anaphase-promoting complex also called cyclosome, is a cell cycle regulated ubiquitin protein ligase (E3) that controls important transitions in mitosis and G1 (reviewed by 1"3 ).
  • E3 cell cycle regulated ubiquitin protein ligase
  • the APC initiates sister chromatide separation by ubiquitinating the anaphase inhibitor securin and it triggers exit from mitosis by ubiquitinating cyclin B, the activating subunit of cyclin-dependent kinase 1 .
  • E1 enzyme ubiquitin activating enzyme
  • E2 may collaborate with several different E3 proteins in creating a protein-ubiquitin conjugate. Together, E2 and the APC assemble multi-ubiquitin chains on substrate proteins and thereby target them for degradation by the 26S proteasome.
  • the activity of the APC is tightly controlled during the cell cycle 2 ' 3 .
  • the APC is activated by phosphorylation and subsequent binding of the activator protein CDC20, whereas the APC is kept active during G1 and in differentiated cell by binding of CDH1 .
  • mitotic activation of the APC is negatively controlled by the MAD2 protein that inhibits APC- CDC20 until spindle assembly has been completed.
  • the APC is composed of at least 1 1 subunits, none of whose structures are known, and most of which are conserved from yeast to man 1#3 . The structures as well as functions are not known for any of these proteins.
  • APC1 1 Only for the RING finger subunit APC1 1 , it has recently been demonstrated that this protein is sufficient to mediate E1 - and E2-dependent ubiquitination reactions in vitro 4,s . APC1 1 may therefore directly participate in substrate ubiquitination mediated by the holo-APC.
  • Another subunit that may have an important role in APC function is the 21 kDa protein APC10/DOC1 .
  • APC10/DOC1 is homologous to domains found in several other putative ubiquitin protein ligases (DOC domains). This protein was first identified in a genetic screen for yeast mutants defective in cyclin proteolysis 6 and was subsequently shown to be a subunit of the APC in budding yeast, fission yeast and vertebrates 6"10 .
  • APC10/DOC1 is highly homologous to a sequence element found in several hypothetical high molecular weight proteins whose domains structure implies that they may also be ubiquitin- protein ligases 9"11 .
  • the APC10/DOC1 homology region is named the DOC domain 10 .
  • Different DOC domain proteins contain combinations of either RING finger or cullin or HECT domains. RING finger domains have been found in many ubiquitin-protein ligases and bind at least in some cases directly to E2 enzymes 12,13 .
  • Cullin domains have been identified in subunits of both the APC and the SCF, a ubiquitin-protein ligase distantly related to the APC, and have been shown to bind to RING finger subunits .
  • HECT domains are found in another type of ubiquitin-protein ligase that covalently binds ubiquitin before transferring it to substrates 15 .
  • DOC domains are found in combination with these three domains implies that they may have a general function in ubiquitination reactions 10 . All of the subunits of APC and especially APC10/DOC1 are therefore a very interesting target for research with the aim to influence ubiquitination reactions and also the anaphase promoting function of APC. It was therefore an object of the present invention to crystallize APC10 and describe its crystal structure which then can be the basis for rational drug design, drug (e.g. inhibitor) screening and further investigations.
  • the subject was solved by providing a crystalline preparation of APC10 with the crystal structure as shown in Fig. 1 .
  • the crystal structure is also shown in the enclosed PDB file.
  • APC10 The crystal structure of human APC10 was ressolved at 1 .6 ⁇ and provides evidence that the C-terminal peptide of APC10 interacts with the APC subunit CDC27. Unexpectedly, the structure of APC10, which is a paradigm for DOC domains, is very similar to the structures of ligand- binding jellyroll domains of several bacterial and eukaryotic proteins.
  • APC10 ⁇ C14 Progressive degradation of the full length human APC10/DOC1 protein during protein purification led us to the preparation and crystallization of the stable fragment, lacking 14 C-terminal residues (APC10 ⁇ C14).
  • the crystal structe of APC10 ⁇ C14 was solved by MAD phasing, using crystals containing the selenomethionine labeled protein and one nickel ion per molecule, originating from the crystallization conditions. Data collection and refinement statistics are shown in Table 1 . An excellent electron density was obtained and used for model building. All residues were resolved in the density except the side chains of the residues Asn127- Lys1 29 and the C-terminal residues Glu1 63-Pro171 .
  • the overall structure of the single domain is dominated by ⁇ -sheets (Fig. 1 a).
  • the central core of APC 10 structure consists of a ⁇ -sandwich where a five stranded antiparallel ⁇ -sheet (strands ⁇ 1 , ⁇ 2, ⁇ 7, ⁇ 4, ⁇ 5) is packed on top of a three stranded antiparallel ⁇ -sheet .(strands ⁇ 3, ⁇ 6, ⁇ 8) thereby exhibiting a "jellyroll" fold. Connections across that side of the sandwich where the N- terminus is located are formed by the loop ⁇ 3- ⁇ 4, including the short a- helix ⁇ 2 , and the loop ⁇ 7- ⁇ 8, containing the short ⁇ -strand ⁇ 7'.
  • this region we refer to this region as the N-terminal loop region.
  • A. remarkably large loop in this region is the ⁇ 1 - ⁇ 2 loop that contains the small ⁇ -sheet ⁇ 1 ' and connects two strands of the same sheet.
  • the opposite side of the sandwich is closed by the loops ⁇ 2- ⁇ 3 and ⁇ 6- ⁇ 7, which we call the C-terminal loop region (Fig. 1 a).
  • Additional structural elements are two short ⁇ -helices, ⁇ 1 as the first N-terminal secondary structure element (Fig. 3b) and the helical extension of the ⁇ 3 strand al, as well as the short ⁇ -sheet formed by the strands ⁇ V and ⁇ 7'.
  • All of these proteins have a galactose-binding-domain-like fold and belong to the discoidin domain family 20 .
  • the common function of these domains is to bind specific ligands.
  • galactose oxidase and sialidase these domains bind to cell surface attached carbohydrate substrates, whereas coagulation factor Va binds to phospholipids in the outer side of the mammalian cell membrane 18,2 °.
  • the N-terminal domain of XRCC1 binds to a complex of broken single-stranded DNA and DNA polymerase ⁇ . All of these structurally related proteins use similar regions for ligand binding which corresponds to the N-terminal loop region in APC10.
  • the N-terminal loop region of APC10 may therefore also represent a ligand-binding region.
  • FIG. 3a Comparison of human APC10 (Fig. 3a) with its orthologues in S. cerevisiae, S. pombe and D. melanogaster 2 show that the N-terminal loop region contains several evolutionary strictly conserved residues (Fig. 3c). They represent one continuous epitope, which implies an involvement of this region in APC10 function. Some residues in the ⁇ 1 - ⁇ 1 ' loop, Ser35, Asn47, Thr52, Trp54, Pro61 and His62, are not only conserved among APC10 orthologues but also in galactose oxidase and sialidase (Fig. 4).
  • Mutation of Ser127 in budding yeast Doc1 p/Apc10p, which corresponds to Ser35 in human APC10, has been found to render the protein temperature sensitive, causing yeast cells to arrest in mitosis with an inactive APC 6 .
  • This mutation could inactivate Doc1 p/Apc10p either by interfering with the binding of a hypothetical ligand, or by deforming the protein backbone since the exposition of the hydrophobic phenyl sidechain to the solvent is energetically unfavorable.
  • the Phe127 mutant would be expected to lack the hydrogen bond of the Ser127 side chain and probably also the second hydrogen bond to Trp146 (Trp54 in human APC10). At higher temperatures the remaining interactions of the loop ⁇ 1 - ⁇ 1 ' to the core may not be sufficient to stabilize the structure, leading to distortion of the whole N-terminal loop region.
  • the C-terminus of APC 10 binds to CDC27/APC3
  • Arg127-Arg185 are not present in APC10 ⁇ C14 used for crystalization, and residues Glu163-Pro171 are disordered in the crystal. Because the C- terminus of APC10 might become stabilized and ordered by interaction with a binding partner, we searched for APC subunits that bind to APC10. Co-infection of insect cells with recombinant baculoviruses encoding different APC subunits (Fig. 4a) as well as in vitro binding experiments using His-APC10 and different GST-tagged subunits expressed in E.coli (data not shown) identified CDC27/APC3 as a candidate binding partner of APC10.
  • CDC27 bound to recombinant GST-APC10 purified from E.coli but not or little to other APC subunits (Fig. 4b, c), suggesting that this interaction is specific.
  • CDC27 specifically co-immunoprecipitated the full length form of APC10, whereas a smaller fragment remained in the supernatant (Fig. 4a). Since this fragment retained the N-terminal tag, it must have been degraded from the C-terminus.
  • CDC27 contains l Otetratrico peptide repeats (TPRs), a motif that is known to be capable of binding elongated peptide stretches 22,23 .
  • TPRs Otetratrico peptide repeats
  • CDC16/APC6 another APC subunit with multiple TPR motifs that interacted with APC10 to a lesser extent in insect cells (data not shown) or in vitro (Fig. 4b), bound to the C-terminal APC10 peptide only weakly (Fig. 4b,c).
  • the crystal structure of human APC10 represents the first atomic structure of a subunit of the APC. It is further a paradigm for the fold of the DOC domain, which has been recognized in several proteins related to ubiquitination reactions 9,1 °. Unexpectedly, within the present invention it has been shown that the DOC domain of APC 10 has a jellyroll fold which is highly similar to ligand binding domains found in several eubacterial and eukaryotic proteins such as galactose oxidase 16 , sialidase 17 , blood coagulation factor Va 18 and CRCC1 19 .
  • APC10 The structural conservation between the ligand binding regions of these proteins and the N-terminal loop region of APC10 suggests that the function of APC10 may be to bind a specific ligand within the holo-APC.
  • This ligand may play an important role in ubiquitination reactions because mutation of an evolutionary conserved Ser residue in the N-terminal loop region of budding yeast Doc1 p/Apc10p renders the protein temperature sensitive and thereby appears to inactivate the holo-APC 6 .
  • This notion is further supported by the observation that DOC domain proteins are particularly conserved in those sequence elements that form the N-terminal loop region in APC10.
  • the hydrophobic patch that we have noticed in the N-terminal loop region of APC10 could have an important role in binding this hypothetical ligand.
  • jellyroll domains have been shown to bind ligands such as sugars, nucleotides, phospholipids or DNA.
  • ligands such as sugars, nucleotides, phospholipids or DNA.
  • the role of the jellyroll domain appears to be to position these ligands towards other catalytically active domains 17 .
  • Protein or peptide ligands of this domain have not yet been identified, but a peptide ligand has been proposed for the jellyroll domain of the receptor tyrosine kinase EphB2 2 ⁇
  • CDC27 contains TPR motifs, which in the TPR proteins Hop and PEX5 have been found to bind to extended N-terminal or C-terminal peptide stretches of their partners, respectively 22,23 .
  • the crystalline preparation of APC10 allowed to gain the herein described crystal structure data.
  • Such crystal structure data can be used for the design or/and the identification of modulators, preferably inhibitors of the APC complex by interacting with APC10, e.g. by inhibiting the interaction between APC10 and CDC27. Such inhibition may affect the assembly of the APC complex and thus also the activity of APC.
  • An alternative mode of action, by which an APC10 inhibitor may exert its effect on APC inhibition, is based on a compound's interaction with the N-terminal loop of the APC10 protein. As mentioned above, a mutation in this region in the homologous yeast protein has been shown to result in temperature sensitive mutants, causing yeast cells to arrest in mitosis 6 .
  • Another subject of the present invention is the use of crystal structure data obtained from a crystalline preparation of APC and described herein in detail for the design and/or identification and/or preparation of modulators, preferably inhibitors of APC.
  • a computer-aided modeling program is used for the design of modulator molecules.
  • a data base is screened for possible inhibitors using the crystal structure data obtained according to the present invention.
  • Modulators can influence the activity of APC10 by interacting therewith via binding, either covalently or non-covalently to APC. Other interactions might also lead to a modulating effect like e.g. association of a modulator molecule and the APC10 subunit via van der Waals forces or hydrogen bonding. Modulator molecules and especially inhibitor molecules acting through any one of the above described mechanisms are encompassed by the invention. Also encompassed are other mechanisms for modulating the activity of the APC 10 unit and finally the APC complex, as long as such modulator molecules are designed, screened for or prepared using the crystal structure data of the present invention.
  • APC 10 with other TPR containing APC subunits and also design and/or identification and/or preparation of inhibitors that interfere with such binding are of interest within the framework of the present invention.
  • a further subject of the present invention are inhibitors that specifically inhibit APC by interacting with or binding to APC10.
  • inhibitors Preferably such inhibitors interact with or bind to sites that either are responsible for CDC27 binding or binding of other TPR-containing APC subunits.
  • the aim of most chemotherapeutic approaches against cancer is to kill rapidly proliferating cells while leaving non-proliferating, differentiated cells unaffected. Since the state of the components regulating the cell cycle is different between proliferating and quiescent cells, these components, one of them being the APC, have been suggested as targets for anti-cancer drugs.
  • the APC has been suggested as a target for chemotherapeutic intervention for the following reasons:
  • the activity of the APC is essential for sister chromatid separation, for the function of the mitotic spindle and for exit from mitosis during cell poliferation. Interfering with this function is expected to prevent tumor cells from completing mitosis.
  • Tumor cells have highly abnormal karyotypes. They undergo anaphase in the presence of chromosomal damage that would prevent activation of the APC in normal cells. Tumor cells are therefore expected to be especially sensitive to drugs that interfere with APC function.
  • APC 10 inhibitors by functioning as APC inhibitors, are expected to arrest cells in metaphase of mitosis; this arrest is expected to subsequently induce apoptotic cell death. Due to this ability, APC 10 inhibitors are drug candidates for the therapy of cancer.
  • an APC10 inhibitor to inhibit tumor cell proliferation
  • primary human tumor cells are incubated with the compound and the inhibition of tumor cell proliferation is tested by conventional methods, e.g. bromo-desoxy-uridine or 3 H incorporation.
  • Compounds that exhibit an anti- proliferative effect in these assays may be further tested in tumor animal models and used for the therapy of tumors.
  • Toxicity and therapeutic efficacy of the APC 10 modulators can be determined by standard pharmaceutical procedures, which include conducting cell culture and animal experiments to determine the IC 50 , LD 50 , ED 50 .
  • the data obtained are used for determining the human dose range, which will also depend on the dosage form (tablets, capsules, aerosol sprays, ampules, etc.) and the administration route (oral, buccal, nasal, paterental or rectal).
  • a pharmaceutical composition containing the compound as the active ingredient can be formulated in conventional manner using one or more physologically active carriers and excipients. Methods for making such formulations can be found in manuals, e.g. "Remington Pharmaceutical Sciences".
  • a still further subject of the present invention is a pharmaceutical composition containing such APC inhibitor and further the use of such inhibitor or pharmaceutical composition containing an inhibitor for prevention or treatment in situations where it is required or beneficial to inhibit anaphase promotion by APC.
  • Yellow residues show sequence similarity between the first three sequences.
  • the secondary structure elements are identical below the alignment.
  • the histone H4 N-terminal peptide was used to exclude unspecific peptide or matrix binding by the HisHA-CDC27 protein.
  • the human APC10 cDNA (accession code AF132794) was cloned into pET1 1 d (Novagen, Germany), resulting in an open reading frame encoding 185 residues with the sequence NH 2 -MATP...RSIR-COOH.
  • the threonine residue at position 2 of human APC10 was changed to alanine to optimize the codon usage for E.coli expression.
  • Overexpression of APC10 in the E.coli strain B834(DE3) (Novagen, Germany) pUBS250 26 yielded inclusion bodies from which APC10 was refolded. The refolded protein was degraded into a stable 19.5 kDa fragment.
  • Diffraction data for the MAD (multiwavelength anomalous diffraction) experiment were collected from a single crystal of the selenomethionide labeled protein at 100K at the MPG beamline BW6 at DORIS (DESY, Hamburg, Germany) using a MAR-CCD detector.
  • Refinement of the structure was carried out with CNS 34 using the high- resolution dataset of native APC10 ⁇ C14.
  • positional and B-factor refinement in CNS and model building with O the initial R factor decreased from 34.4% (R fre ⁇ 36.8) to 26.4% (R fre ⁇ 30.1 ) and after the introduction of 1 57 solvent molecules and one nickel ion to 20.9% (R free 24.4%). Since the side chains of residues 1 27-1 29 are not resolved in the density their occupancy was set to 0 during refinement.
  • Example 3 Binding assays Recombinant baculoviruses for human APC subunits containing N-terminal GST- or 10xHis-HA-tags were generated using the Bac-to-Bac system (Life Technologies) . Lysates of infected Hi5 cells were used for in vitro binding assays. Bacterial expression of full length APC10 with an N-terminal GST- tag (GST-APC10) has been described 4 , and GST-APC10 lacking the 23 C- terminal residues (GST-APC10 ⁇ C23) was produced analogously.
  • Detection was performed with monoclonal anti-HA 12CA5 (own production) or anti-6His polyclonal antibodies H-15 (Santa Cruz).
  • purified APC10 ⁇ C14 was coupled to CNBr-activated Sepharose 4 Fast Flow (Pharmacia) and incubated with lysates containing APC subunits.

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Abstract

La présente invention concerne la structure cristalline de la sous-unité APC10/DOC1, une sous-unité du complexe de promotion de l'anaphase (APC) chez l'homme. L'invention concerne également l'utilisation de la structure cristalline de l'APC10/DOC1 de façon à obtenir des données sur la structure cristalline permettant une conception rationnelle de médicaments et la recherche systématique d'inhibiteurs de l'APC. L'invention concerne enfin des compositions pharmaceutiques contenant de tels inhibiteurs ainsi que l'utilisation de ces inhibiteurs pour prévenir l'induction de l'anaphase.
PCT/EP2002/006909 2001-06-22 2002-06-21 Structure cristalline de la sous-unite apc10/doc1 du complexe de promotion de l'anaphase chez l'homme Ceased WO2003000726A1 (fr)

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1167539A1 (fr) * 2000-06-29 2002-01-02 Boehringer Ingelheim International GmbH Méthodes pour l'identification des inhibiteurs des complexes promoteurs de l'anaphase

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1167539A1 (fr) * 2000-06-29 2002-01-02 Boehringer Ingelheim International GmbH Méthodes pour l'identification des inhibiteurs des complexes promoteurs de l'anaphase

Non-Patent Citations (9)

* Cited by examiner, † Cited by third party
Title
AU SHANNON W N ET AL: "Implications for the ubiquitination reaction of the anaphase-promoting complex from the crystal structure of the Doc1/Apc10 subunit.", JOURNAL OF MOLECULAR BIOLOGY, vol. 316, no. 4, 2002, 1 March, 2002, pages 955 - 968, XP002218319, ISSN: 0022-2836 *
DATABASE PDB [online] 31 January 1994 (1994-01-31), ITO, N. ET AL.: "Galactose Oxidase (E.C. 1.1.3.9) (pH 4.5)", XP002218321, retrieved from WWW.RCSB.ORG Database accession no. 1GOF *
EISENSTEIN E ET AL: "BIOLOGICAL FUNCTION MADE CRYSTAL CLEAR - ANNOTATION OF HYPOTHETICAL PROTEINS VIA STRUCTURAL GENOMICS", CURRENT OPINION IN BIOTECHNOLOGY, LONDON, GB, vol. 1, no. 11, February 2000 (2000-02-01), pages 25 - 30, XP001062869, ISSN: 0958-1669 *
FARBER G K: "NEW APPROACHES TO RATIONAL DRUG DESIGN", PHARMACOLOGY AND THERAPEUTICS, ELSEVIER, GB, vol. 3, no. 84, December 1999 (1999-12-01), pages 327 - 332, XP001062868, ISSN: 0163-7258 *
GIEFFERS CHRISTIAN ET AL: "Three-dimensional structure of the anaphase-promoting complex.", MOLECULAR CELL, vol. 7, no. 4, April 2001 (2001-04-01), pages 907 - 913, XP002218320, ISSN: 1097-2765 *
GROSSBERGER RUPERT ET AL: "Characterization of the DOC1/APC10 subunit of the yeast and the human anaphase-promoting complex.", JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 274, no. 20, 14 May 1999 (1999-05-14), pages 14500 - 14507, XP002218317, ISSN: 0021-9258 *
KURASAWA YASUHIRO ET AL: "Identification of human APC10/Doc1 as a subunit of anaphase promoting complex.", ONCOGENE, vol. 18, no. 37, pages 5131 - 5137, XP002218316, ISSN: 0950-9232 *
SKELLY JANE V ET AL: "Overexpression, isolation, and crystallization of proteins.", METHODS IN MOLECULAR BIOLOGY, vol. 56, 1996, 1996 Humana Press Inc. Suite 808, 999 Riverview Drive, Totowa, New Jersey 07512, USA, pages 23 - 53, XP002218318, ISBN: 0-89603-259-0 *
WENDT K S ET AL: "Crystal structure of the APC10/DOC1 subunit of the human anaphase-promoting complex.", NATURE STRUCTURAL BIOLOGY. UNITED STATES SEP 2001, vol. 8, no. 9, September 2001 (2001-09-01), pages 784 - 788, XP001107138, ISSN: 1072-8368 *

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