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WO2002022862A2 - Methodes d'identification de modulateurs d'interactions de proteines - Google Patents

Methodes d'identification de modulateurs d'interactions de proteines Download PDF

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Publication number
WO2002022862A2
WO2002022862A2 PCT/GB2001/004094 GB0104094W WO0222862A2 WO 2002022862 A2 WO2002022862 A2 WO 2002022862A2 GB 0104094 W GB0104094 W GB 0104094W WO 0222862 A2 WO0222862 A2 WO 0222862A2
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Prior art keywords
ilk
agent
pkb
activity
psl
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WO2002022862A3 (fr
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Ian Donald Hiles
Christine Ellis
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Glaxo Group Ltd
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Glaxo Group Ltd
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Priority to AU2001286091A priority Critical patent/AU2001286091A1/en
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Publication of WO2002022862A3 publication Critical patent/WO2002022862A3/fr
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • C12Q1/37—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving peptidase or proteinase
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/48—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase
    • C12Q1/485—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase involving kinase
    • 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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/02—Screening involving studying the effect of compounds C on the interaction between interacting molecules A and B (e.g. A = enzyme and B = substrate for A, or A = receptor and B = ligand for the receptor)

Definitions

  • This invention relates to methods for identifying agents capable of modulating protein interactions. It further relates to the use of such agents in treating or preventing conditions associated with apoptosis or Alzheimer's disease.
  • PKB Protein kinase B regulates cellular activities as diverse as glycogen metabolism and apoptosis.
  • PKB exerts its anti-apoptotic effects in cells via the inhibition of key enzymes including glycogen synthase kinase-3 (GSK3) (resulting in reduced tau phosphorylation and enhanced beta-catenin-LEF-1 transcriptional activity), BAD, caspase 9 and the forkhead transcription factor FKHLR1.
  • GSK3 glycogen synthase kinase-3
  • BAD caspase 9
  • FKHLR1 forkhead transcription factor
  • PKB regulation is known to be influenced by many factors, for example insulin, growth factors and integrins. Indeed, PKB activation is likely to represent a convergence point for the integration of signals from growth factors and the extracellular matrix.
  • the full activation of PKB alpha requires 3-phosphoinositides and dual phosphorylation on threonine-308 (T308) and serine-473 (S473).
  • Corresponding activation sites in beta and gamma forms are T309 and S474 for beta and S305 and T472 for gamma.
  • T308 is found within the activation loop of the kinase domain and S473 in the c-terminal tail.
  • PDK1 3- phosphoinositide dependent-kinase 1
  • PDK2 3-phosphoinositide dependent- kinase 2
  • autophosphorylation may be the mechanism for S473 phosphorylation on PKB (Toker and Newton, J Biol. Chern. 275: 8271-
  • MPKAP kinase 2 phosphorylates S473 in vitro, as does PDK1 bound to protein kinase-C related kinase-2 (PRK2) or a C-terminal peptide thereof.
  • Integrin-linked kinase (ILK) has also been suggested as a putative S473 kinase. ILK was first identified as a serine/threonine-specific protein kinase able to associate stably with beta-integrins (as demonstrated by both yeast 2 hybrid and co- precipitation approaches).
  • ILK-2 Janji et al., Oncogene 19:3069-3077, 2000
  • ILK has been shown to regulate a PKB-mediated cell survival pathway. Further studies have suggested that ILK functions as an adaptor to recruit a S473 kinase or phosphatase (Lynch et al, Oncogene 18: 8024-32, 1999).
  • Another putative regulator of PKB is presenilin-1 (PSl) (Weihl et al., Journal ofNeuroscience 19(13):5360-69, 1999).
  • PSl is an integral membrane protein required for the production of beta-amyloid from amyloid precursor protein (APP).
  • AD Alzheimer's disease
  • ⁇ - and gamma ( ⁇ -) secretases are cleaved from amyloid precursor protein by the action of beta ( ⁇ -) and gamma ( ⁇ -) secretases at two sites; proximal to position 1 (due to the action of beta secretase) and distal to residues 40, 42 or 43 (due to the action of gamma secretase).
  • AD- causing presenilin 1 mutants cause a change in processing so that cleavage at residues 42 and 43 is favoured over processing at residue 40.
  • AD-linked preseniling 1 mutations may alter the ratio of beta-amyloid 1-40: 1-42/43 in favour of the latter, without alteration of the amount of beta amyloid processing.
  • mutations in amyloid precursor protein can enhance its proteolytic cleavage and therefore increase amyloid-beta production whilst mutations in presenilin-1 (PSl) or presenilin-2 (PS2) can alter ⁇ -secretase activity and production of amyloid-beta.
  • PSl presenilin-1
  • PS2 presenilin-2
  • ⁇ -secretase activity may be an intrinsic property of PS-1 and PS-2.
  • PS-1 mutant proteins In addition to the effects of AD- associated PS-1 mutant proteins on ⁇ -secretase activity, a recent report (Weihl et al., 1999) showed that they also inhibit PKB activity, concomitant with increased neuronal apoptosis.
  • Other reports have noted that PSl associates with downstream targets of PKB activity, in particular, glycogen synthase kinase-3 ⁇ (GSK-3 ⁇ ) and ⁇ - catenin. It is suggested that this may be the means by which PS 1 influences PKB activity.
  • ILK integrin-linked kinase
  • PSl presenilin-1
  • PBB protein kinase B
  • modulators of the interaction of ILK with PSl represent suitable candidates for the treatment of conditions associated with apoptosis and Alzheimer's disease.
  • the invention provides a method for identifying an agent that modulates the interaction between integrin-linked kinase (ILK) and presenilin-1 (PSl) comprising:
  • step (d) determining whether the test agent is capable of modulating the interaction between the first and second components; thereby to determine whether the test agent is a modulator of the interaction between ILK and PS 1.
  • step (d) comprises determining whether the test agent is capable of inhibiting the interaction between ILK and PSl, thereby to determine whether the test agent is a inhibitor of the interaction between ILK and PSl .
  • the method further comprises combining the agent thus identified with a pharmaceutically acceptable carrier or diluent, thereby to produce a pharmaceutical composition.
  • the invention also provides a method for identifying an agent that modulates the activity of protein kinase B (PKB) or ⁇ -secretase comprising:
  • step (iii) comprises determining whether the test agent is capable of stimulating the activity of PKB or ⁇ -secretase, thereby to determine whether the test agent is a stimulator of PKB or ⁇ -secretase activity.
  • step (iv) comprises determining whether the test agent is capable of inhibiting the activity of PKB or ⁇ -secretase, thereby to determine whether the test agent is a inhibitor of PKB of ⁇ -secretase activity.
  • An agent that is identified can be formulated with a pharmaceutically acceptable carrier or diluent, thereby to produce a pharmaceutical composition.
  • test kit for the identification of an agent that modulates the interaction between ILK and PSl wherein the kit comprises:
  • test kit for the identification of an agent that modulates the activity of PKB or ⁇ -secretase, wherein the kit comprises:
  • PKB a condition associated with the activity of ⁇ -secretase, a condition associated with apoptosis, or Alzheimer's disease
  • administering to the host an effective amount of an agent of the invention; a pharmaceutical composition comprising a pharmaceutically acceptable carrier or diluent and an agent of the invention; an agent of the invention for use in a method of treatment of the human or animal body by therapy, preferably a condition associated with the interaction between ILK and PSl, a condition associated with the activity of PKB, a condition associated with the activity of ⁇ -secretase, a condition associated with apoptosis, or Alzheimer's disease; and use of an agent of the invention in the manufacture of a medicament for use in the treatment of a condition associated with the interaction between ILK and PSl, a condition associated with the activity of PKB, a condition associated with the activity of ⁇ -secretase, a condition associated with apoptosis, or Alzheimer's disease.
  • Panels A-C COSl cells (lanes 2-4) were transiently co transfected with ILK and PSl (lane 2), PSl M146N (lane 3) or PSl C263R. COSl cells in lane 1 were mock transfected.
  • Figure 1 A and B RIPA extracts were prepared, fractionated by SDS-PAGE and subjected to western blotting using antisera specific for ILK (AB29)
  • Panel A cells were lysed in ILK lysis buffer and immunoprecipitates prepared with an ILK specific antisera. Samples were fractionated by SDS-PAGE and subjected to western blotting using antisera specific for PSl.
  • Panels D-F COSl cells (lanes 2-5) were transiently co transfected with ILK and PSl N-EYFP (lane 2), PSl C-EYFP (lane 3), PSl M146N ⁇ -EYFP (lane 4) or PSl C-EYFP (lane 5). COSl cells in lane 1 were mock transfected.
  • Panels D and F Cells were lysed in ILK lysis buffer and immunoprecipitates prepared with antisera specific for either PSl (D) or ILK (F). Samples were fractionated by SDS-PAGE and subjected to western blotting using antisera specific for PSl.
  • Panel E RIPA extracts were prepared, fractionated by SDS-PAGE and subjected to western blotting using antisera specific for ILK (AB29).
  • Panel A COSl cells (lanes 2-5) were transiently co transfected with ILK and PSl ⁇ -EYFP (lane 2), PSl C-EYFP (lane 3), PSl M146N ⁇ -EYFP (lane 4) or PSl C-EYFP (lane 5).
  • Panel B COSl cells were transiently co-transfected with ILK and PSl delta 9.
  • Panel C lane 1 Mouse hippocampal neurons. Cells were lysed in ILK lysis buffer and immunoprecipitates prepared with antisera specific for PSl.
  • Panel C lanes 2-4 anti-PS- 1 antisera 1 Dl (lane 2), 0.5 Dl (lane 2) or 0.2 Dl (lane 3). Samples were fractionated by SDS-PAGE and subjected to western blotting using antisera specific for ILK.
  • (A-C) COSl cells were transiently co-transfected with PSl ⁇ -EYFP and ⁇ - Flag ILK (lane 1), C-Flag-ILK K->M (lane 2) or C-Flag ILK (lane 3).
  • Panel D COSl cells were transiently co-transfected with PSl ⁇ -EYFP and wild-type ILK (lane 1), FEF ILK (lane 2) or ILK K->M (lane 3).
  • Panel A RIPA extracts were prepared, fractionated by SDS-PAGE and subjected to western blotting using antisera specific for Flag.
  • Panels B-D Cells were lysed in ILK lysis buffer and immunoprecipitates prepared with antisera specific for PSl . Samples were fractionated by SDS-PAGE and subjected to western blotting using antisera specific for PSl (B), Flag (C) or ILK (D).
  • D D
  • Lanes 2,5) or Myc-Dvl2 Lanes 3,6.
  • COSl cells in lanes 1 and 4 were mock transfected.
  • Lanes 1-3 RIPA extracts were prepared, fractionated by SDS-PAGE and subjected to western blotting using antisera specific for Myc tag.
  • Lanes 4-6 cells were lysed in ILK lysis buffer and immunoprecipitates prepared with ILK specific antisera. Samples were fractionated by SDS-PAGE and subjected to western blotting using antisera specific for Myc tag.
  • COSl cells were transiently co transfected with PSl N-EYFP and empty vector (lane 1), P85 subunit of PI3-kinase (lane 2) ILK (lane 3), COS cells singly transiently transfected with ILK (lane 4), mock transfected (lane 5) or transfected with P85 subunit of PI3-kinase (lane 6).
  • Cells were lysed in ILK lysis buffer and immunoprecipitates prepared with ILK specific antisera (lanes 1-5) or antisera raised against the P85 subunit of PI3-kinase (lane 6). Samples were fractionated by SDS- PAGE and subjected to western blotting using antisera specific for PSl.
  • ILK integrin-linked kinase
  • PSl presenilin-1
  • compositions or the integrin-linked kinase signalling pathway may, therefore, provide a means to modulate amyloid-beta production and/or to up-regulate specifically the protein kinase B cell survival pathway in neuronal cells susceptible to apoptosis in AD.
  • the invention provides methods for identifying a modulator of the interaction between ILK and PSl. ILK or a functional variant thereof is contacted with PS 1 or a functional variant thereof in the presence of the test agent under conditions that, in the absence of the test agent, would permit the two components to interact. The capability of the test agent to modulate the interaction between ILK or the ILK variant and PSl or the PSl variant is then determined.
  • ILK or a functional variant thereof is provided as a first component.
  • ILK as used herein refers to either ILK, or ILK-2.
  • the amino acid sequence of ILK is set out in SEQ ID NO: 1.
  • the amino acid sequence of ILK-2 is provided in SEQ ID NO: 2.
  • the second component comprises PSl or a functional variant thereof.
  • the amino acid sequence of PSl is set out in SEQ ID NO: 3.
  • the second component comprises PS2 or a function variant thereof instead of PS 1.
  • the amino acid seqeunce of PS2 is set out in SEQ ID NO: 4.
  • the invention also provides methods for identifying a modulator of the activity of protein kinase B (PKB) and ⁇ -sectretase.
  • PKB protein kinase B
  • a system comprising ILK, PSl and either PKB or ⁇ -secretase or functional variants of any of these is contacted with, as a test agent, a modulator of the interaction between ILK and PSl identifiable by a method of the invention under conditions that, in the absence of the test agent, would permit PKB or ⁇ -secretase activity to be regulated by the interaction between ILK and PSl .
  • the capability of the test agent to modulate the activity of PKB or ⁇ - secretase is then determined.
  • Systems of this method comprise ILK, PSl and either
  • the system may be in vivo or in vitro.
  • An in vivo system may comprise a cell, a culture of cells or a whole organism. Some or all of the components of the in vivo system may be expressed by the system or provided by other means. Where the system expresses components, some or all of the expressed components may be expressed naturally or recombinantly within the system.
  • An in vitro system may comprise recombinant or chemically synthesised components.
  • PKB as used herein refers to PKB-alpha, PKB-beta or PKB-gamma.
  • the amino acid sequence of PKB-alpha is set out in SEQ ID NO: 5.
  • the amino acid sequence of PKB-beta is set out in SEQ ID NO: 6.
  • the amino acid sequence of PKB-gamma is set out in SEQ ID NO: 7.
  • Test agents which show activity in assays such as those described above can be tested in in vivo systems, such as an animal model of apoptosis and/or
  • Alzheimer's disease Alternatively the activity of such test agents on apoptosis may be tested using in vitro assays, e.g. to look at induction of neuronal apoptosis (Weihl et al, 1999). Thus, candidate agents could be tested for their ability to modulate apoptosis and/or Alzheimer's disease, thereby to identify a modulator of apoptosis and/or Alzheimer's disease. Therefore the invention also provides a methods for identifying a modulator of apoptosis and/or Alzheimer's disease.
  • a suitable method of the invention comprises: providing, as a first component, a system susceptible to apoptosis or amyloid-beta deposition; providing, as a second component, an agent identifiable as a modulator of the interaction between ILK and PSl or the activity of PKB or ⁇ -secretase; contacting the first and second components under conditions that, in the absence of the second component, would permit apoptosis or amyloid- beta deposition; and determining whether the test agent is capable of modulating apoptosis or amyloid-beta deposition.
  • the skilled person can thereby readily determine whether the test agent is a modulator of apoptosis or Alzheimer's disease.
  • Systems of this method are typically cellular. Usually the cellular system will be a cell culture systems, or an animal system, e.g. mice or rats.
  • a functional variant is a polypeptide which has a sequence similar to, and which retains the activity of, the polypeptide sequence from which it is derived (the polypeptide sequence from which the functional variant is derived being herein referred to as the 'wild-type').
  • the wild-type refers to the sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2.
  • the wild- type refers to the sequence set forth in SEQ ID NO: 3.
  • PS2 the wild- type refers to the sequence set forth in SEQ ID NO: 4.
  • the wild-type refers to the sequence set forth in SEQ ID NO: 5.
  • the wild-type refers to the sequence set forth in SEQ ID NO: 6.
  • the wild type refers to the sequence set forth in SEQ ID NO: 7
  • the binding activity of a functional variant with respect to the binding of a target may be substantially the same as that of the wild-type.
  • the target is a target which is specifically bound by the wild-type polypeptide in vivo.
  • the preferred target is PS 1.
  • the preferred target is ILK.
  • preferred targets are GSK3, BAD, > 9 or FKHLR1.
  • the preferred target is amyloid precursor protein (AAP).
  • AAP amyloid precursor protein
  • other preferred targets include b-catenin and GSK3 (Nan Gassen et al, 2000, Neurobiology of Disease, 7:135-151).
  • the binding activity of a functional variant with respect to the binding of a target may be greater or less than that of the wild-type.
  • a functional variant may have at least 90% activity, at least 80% activity or at least 70% activity of the wild-type with respect to its ability to bind its target.
  • a functional variant may comprise, for example, a fragment of the wild-type which binds the target, or a polypeptide which comprises wild-type sequences which correspond to target binding sites and elsewhere comprises non- wild-type sequences.
  • a functional variant typically comprises a sequence substantially similar to the wild-type.
  • a functional variant will generally have at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98 or at least 99% sequence identity to the wild-type, calculated over the full length of those sequences.
  • the UWGCG Package provides the BESTFIT program which can be used to calculate identity (for example used on its default settings) (Devereux et al (1984) Nucleic Acids Research 12, p387-395).
  • the PILEUP and BLAST algorithms can be used to calculate identity or line up sequences (typically on their default settings), for example as described in Altschul S. F.
  • a functional variant may be a naturally occurring sequence, for example an allelic variant.
  • An allelic variant will generally be of human or non-human mammal, for example bovine or porcine, origin.
  • a functional variant may be a non-naturally occurring sequence.
  • a non-naturally occurring functional variant may be a modified version of the wild-type, obtained by, for example, amino acid substitution or deletion. Up to 1, up to 5, up to 10, up to 50 or up to 100 amino acid substitutions or deletions, for example, may be made. Typically, the substitutions will be conservative substitutions, for example according to the following Table. Amino acids in the same block in the second column and preferably in the same line in the third column may be substituted for each other. Deletions are preferably deletions of amino acids from one or both ends of the wild-type sequence. Alternatively, deletions are of regions not involved in the binding of the target.
  • the wild-type or a functional variant thereof may be fused to additional heterologous polypeptide sequence, termed a fusion partner, to produce a fusion polypeptide.
  • a fusion partner may be provided at, for example, one or both termini of the wild-type or a functional variant thereof.
  • a fusion partner sequence may perform any function known in the art. Typically, it may be added for the purpose of providing a carrier polypeptide, by which the wild-type or a functional variant thereof can be, for example, affixed to a label, solid matrix or carrier.
  • the fusion partner may be a fluorescent protein such as GFP or a variant thereof.
  • the first and second components for use in a method of the invention are fusion proteins comprising the sequence of different fluorescent proteins that are suitable for use in protein interaction assay techniques known in the art such as Fluorescence Resonance Energy Transfer (FRET) (Latif and FRET).
  • FRET Fluorescence Resonance Energy Transfer
  • a component for use in a method of the invention may be in the form of a fusion polypeptide which comprises heterologous sequences.
  • fusion polypeptides may be easily and cheaply produced in recombinant cell lines, for example recombinant bacterial or insect cell lines. Fusion polypeptides may be expressed at higher levels than the wild-type or a functional variant thereof. Typically this is due to increased translation of the encoding RNA or decreased degradation.
  • fusion polypeptides may be easy to identify and isolate.
  • fusion polypeptides will comprise a polypeptide sequence as described above and a carrier or linker sequence.
  • the carrier or linker sequence will typically be derived from a non-human, preferably a non-mammalian source, for example a bacterial source. This is to minimize the occurrence of non-specific interactions between heterologous sequences in the fusion polypeptide and the target of the wild-type.
  • Polypeptides may be modified by, for example, addition of histidine residues, a T7 tag or glutathione S-transferase, to assist in their isolation.
  • the fusion partner may, for example, promote secretion of the polypeptide from a cell or target expression of the polypeptide to a particular subcellular location, such as the cell membrane, an organelle, an organellar membrane or the nucleus. Sequences that target polypeptides to these locations are well known in the art. Fusion partners can be from 1 to 400 amino acids in length or more typically from 5 to 200 residues in length.
  • the polypeptide may be linked to a fusion partner directly or via an intervening linker sequence. Typical amino acid residues used for linking are tyrosine, cysteine, lysine, glutamic acid or aspartic acid.
  • Suitable polypeptides for use as a first component may be chemically modified, for example, post-translationally modified. For example they may be glycosylated or comprise modified amino acid residues. Polypeptides can be in a variety of forms of polypeptide derivatives, including amides and conjugates with polypeptides.
  • Chemically modified polypeptides also include those having one or more residues chemically derivatized by reaction of a functional side group.
  • Such derivatized side groups include those which have been derivatized to form amine hydrochlorides, p-toluene sulfonyl groups, carbobenzoxy groups, t-butyloxycarbonyl groups, chloroacetyl groups and formyl groups.
  • Free carboxyl groups may be derivatized to form salts, methyl and ethyl esters or other types of esters or hydrazides.
  • Free hydroxyl groups may be derivatized to form O-acyl or O-alkyl derivatives.
  • the imidazole nitrogen of histidine may be derivatized to form N-im- benzylhistidine.
  • chemically modified polypeptides are those polypeptides which contain one or more naturally occurring amino acids derivatives of the twenty standard amino acids.
  • 4-hydroxyproline may be substituted for proline or homoserine may be substituted for serine.
  • the wild-type polypeptide or a functional variant thereof and/or other polypeptides used as part of a component in a method of the invention may carry a revealing label.
  • Suitable labels include radioisotopes such as 32 P or 35 S, fluorescent or luminescent labels, enzyme labels, or other protein labels such as biotin.
  • the wild-type polypeptide or a functional variant thereof and/or other polypeptides used as part of a first component may be expressed using recombinant DNA techniques.
  • suitable polypeptides may be expressed in bacterial, yeast or insect cell lines (see, for example, Munger et al., 1998, Molecular Biology of the Cell, 9, 2627-2638).
  • suitable polypeptides may be isolated biochemically from any suitable tissue.
  • polypeptides may be chemically synthesized.
  • Synthetic techniques such as a solid-phase Merrifield-type synthesis, may be preferred for reasons of purity, antigenic specificity, freedom from unwanted side products and ease of production. Suitable techniques for solid-phase peptide synthesis are well known to those skilled in the art (see for example, Merrifield et al, 1969, Adv.
  • solid-phase synthesis methods comprise the sequential addition of one or more amino acid residues or suitably protected amino acid residues to a growing peptide chain.
  • Polypeptides for use as a first component in a method of the invention may be linear or cyclic.
  • a linear polypeptide may be cyclised according to any suitable method (see for example Zimmer et al., 1992, Peptides, pp.393-394, ESCOM Science Publishers, BN., 1993 and Gurrath et al, 1992, Eur. J. Biochem., 210, 911- 921).
  • tertbutoxycarbonyl protected polypeptide methyl ester is dissolved in methanol and sodium hydroxide are added and the admixture is reacted at 20°C to hydrolytically remove the methyl ester protecting group.
  • the tertbutoxycarbonyl protecting group is extracted with ethyl acetate from acidified aqueous solvent.
  • the tertbutoxycarbonyl protecting group is then removed under mildly acidic conditions in dioxane cosolvent.
  • the unprotected linear peptide with free amino acid and carboxy termini so obtained is converted to its corresponding cyclic polypeptide by reacting a dilute solution of the linear polypeptide in a mixture of dimethylformamide with dicyclohexylcarboiimide in the presence of 1 -hydroxy benzotriazole and ⁇ -methylmorpholine.
  • the resultant cyclic polypeptide is purified by chromatography.
  • a two component assay can be carried out according to any suitable protocol.
  • the assay is adapted so that it can be carried out in a single reaction vessel and more preferably can be carried out in a single well of a plastics microtitre plate and thus can be adapted for high through-put screening.
  • a modulator of the interaction between ILK and PSl is one which causes a change in the degree of interaction between those proteins.
  • the modulator will inhibit or stimulate the interaction between ILK and PS 1.
  • the modulator is an inhibitor and causes the interaction between ILK and PSl to be reduced or substantially eliminated, as compared to the degree of interaction between the two, in the absence of the agent.
  • Preferred agents are those which inhibit the ILK/PS 1 interaction by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or at least 99% at a concentration of the agent of 1 ⁇ gml "1 , 10 ⁇ gml “1 , 100 ⁇ gml "1 , 500 ⁇ gml "1 , 1 mgml "1 , 10 mgml "1 , lOOmg ml "1 .
  • the percentage inhibition represents the percentage decrease in the interaction between ILK/PS 1 in a comparison of assays in the presence and absence of the test agent. Any combination of the above mentioned degrees of percentage inhibition and concentration of modulator may be used to define an agent of the invention, with greater inhibition at lower concentrations being preferred.
  • a modulator of the activity of PKB or ⁇ -secretase is one which causes a change in the degree of activity of those proteins. Typically the modulator will inhibit or stimulate the activity of PKB or ⁇ -secretase. In one embodiment, the modulator causes stimulation of PKB activity. In another embodiment the modulator causes inhibition of ⁇ -secretase activity.
  • preferred agents are those which stimulate the activity of PKB by up to 50%, up to 80%, up to 100%, up to 200%, up to 400%, up to 800%, up to 1000% at a concentration of the agent of 1 ⁇ gml "1 , 10 ⁇ gml “1 , 100 ⁇ gml “1 , 500 ⁇ gml “1 , 1 mgml “1 , 10 mgml “1 , lOOmg ml "1 .
  • the percentage stimulation in activity represents the percentage by which activity of PKB is stimulated in a comparison of assays in the presence and absence of the test agent.
  • preferred agents are those which reduce the activity of ⁇ - secretase to at most 90%, 80%, 70%, 60%, 50%, 40%, 30% or 20%, more preferably 10%, yet more preferably to 5%, 2%, 1% or 0% of the activity of ⁇ -secretase in the absence of the agent, at a concentration of the agent of 1 ⁇ gml "1 , 10 ⁇ gml “1 , 100 ⁇ gml “1 , 500 ⁇ gml "1 , 1 mgml "1 , 10 mgml "1 , lOOmg ml "1 .
  • any combination of the above mentioned degrees of percentage stimulation or inhibition of PKB or ⁇ - secretase and concentration of modulator may be used to define an agent of the invention, with greater stimulation of PKB or greater inhibition of ⁇ -secretase at lower concentrations being preferred.
  • a modulator of ⁇ - secretase activity will change the processing of beta-amyloid, typically in favour of beta-amyloid 1-40 over 1-42/43.
  • a modulator of apoptosis is one which causes a change in the rate of apoptosis.
  • the modulator will inhibit or stimulate apoptosis.
  • the modulator is an inhibitor and causes of the rate of apoptosis to be to be reduced or substantially eliminated, as compared to the rate of apoptosis in the absence of the agent.
  • Preferred agents are those which inhibit the rate of apoptosis by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or at least 99% at a concentration of the agent of 1 ⁇ gml "1 , 10 ⁇ gml “1 , 100 ⁇ gml "1 , 500 ⁇ gml "1 , 1 mgml "1 , 10 mgml "1 , lOOmg ml "1 . Any combination of the above mentioned degrees of percentage inhibition of apoptosis and concentration of modulator may be used to define an agent of the invention, with greater inhibition at lower concentrations being preferred.
  • a modulator of Alzheimer's disease is one which causes a change in the rate of amyloid-beta deposition.
  • the modulator will inhibit or stimulate amyloid-beta deposition.
  • the modulator is an inhibitor and causes of the rate of amyloid-beta deposition to be to be reduced or substantially eliminated, as compared to the rate of amyloid-beta deposition in the absence of the agent.
  • Preferred agents are those which inhibit the rate of amyloid-beta deposition by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or at least 99% at a concentration of the agent of 1 ⁇ gml "1 , 10 ⁇ gml “1 , 100 ⁇ gml "1 , 500 ⁇ gml "1 , 1 mgml "1 , 10 mgml "1 , lOOmg ml "1 . Any combination of the above mentioned degrees of percentage inhibition of amyloid-beta deposition and concentration of modulator may be used to define an agent of the invention, with greater inhibition at lower concentrations being preferred.
  • test agent The effect of a test agent on the interaction between ILK and PSl may be determined by techniques common in the art. Typically, tests are performed in vivo or in vitro. Examples include FRET, homogeneous time resolved fluorescence (HTRF), scintillation proximity assay (SPA), ELISA and other types of protein- protein interaction assays.
  • FRET homogeneous time resolved fluorescence
  • SPA scintillation proximity assay
  • ELISA ELISA
  • HTRF is based on the non-radiative energy transfer from a donor, the rare earth metal Europium, to an acceptor, allophycocyanin (APC).
  • Europium absorbs excitation light at 337 nm and emits a long-lived (time-resolved) fluorescence at 620 nm with a life time in the ⁇ second range. This is longer than conventional fluorophores which have fluorescence lifetimes of nanoseconds (e.g. fluorescein emits over 4-5 nsecs.). If the acceptor molecule is in close proximity to the donor there is energy transfer and the APC emits a specific time-resolved fluorescent signal at 665 nm.
  • HTRF has the advantage of being non-isotopic. Another advantage of HTRF comes from the time-resolved nature of the emitted fluorescence, which means that the first part of the signal which will contain most of the background short lifetime signal can be "gated" out.
  • SPA is based on the conversion of low energy Beta-emissions to light. These low energy electrons are easily absorbed by the medium unless the isotope is in very close proximity (i.e. bound) to the SPA bead. Thus bound and free label do not need to be separated in order to detect a signal, and so SPA is a homogeneous assay method.
  • the emitted light can be measured by scintillation counting, e.g. with instruments capable of reading 96-well microtitre plates
  • a first component polypeptide (either ILK or PSl or functional variants thereof) is immobilised on the walls of a suitable vessel, in particular the well of a plastics microtitre plate.
  • a second component (being PSl where the first component is ILK, and being ILK where the first component is PSl or functional variants thereof), produced, for example, chemically or recombinantly is simply added to the assay vessel. Binding of the second component to the first component can be followed by the labelling of the second component, for example with a radioactive label or a fluorescent label.
  • This type of approach is suitable for an ELISA, which typically involves immobilising one component e.g. ILK with an anti-ILK antibody or tagged ILK with an antibody to the tag. This is then contacted with a solution containing PSl or tagged PSl and binding allowed to occur (in the presence of a test agent if required). After washing, bound PSl can then be detected by an appropriate antibody (coupled to an easily assayed enzyme e.g. horse radish peroxidase) specific for either PSl or the tag present on PS 1.
  • an appropriate antibody coupled to an easily assayed enzyme e.g. horse radish peroxidase
  • Other suitable techniques include immunoprecipitation, western blotting in cell lines or by assay of a functional readout such as a change in amyloid production or change in gamma secretase activity in an in vitro assay.
  • reaction mixture may be necessary to add further components to the reaction mixture in order to stimulate interaction between ILK and PSl, for example, in order to change the ion, carbohydrate or protein concentration or alter the pH of the mixture.
  • the reaction mixture comprising cell cytosol.
  • suitable control experiments may be carried out.
  • the cell adhesion assay may be run without the test agent present.
  • control cells expressing polypeptides other than the second component may be used to distinguish between specific reactions between the first and second components and non-specific reactions between the first component and other surface proteins of the cells expressing the second component.
  • modulators of the interaction between ILK and PSl can be identified by a rational design approach.
  • ILK and PSl may be used in structural analysis methods for the design of agents which are capable of disrupting the interaction between these two polypeptides.
  • Narious structural analysis methods for drug-design are known in the art, including, for example, molecular modelling, two- dimensional nuclear magnetic resonance (2-D ⁇ MR) analysis and x-ray crystallography.
  • Computer-based methods which permit the identification of compounds with a desired molecular structure may be used to identify compounds whose structure is similar to all or a part of an agent of interest. Thus, compounds whose structure is similar to the polypeptides of the invention may be identified and such compounds may be capable of disrupting the interaction between ILK and PSl .
  • Such computer- based methods fall into two-broad classes: database methods and de novo design methods.
  • database methods the compound of interest is compared to all compounds present in a database of chemical structures and compounds whose structure is in some way similar to the agent of interest are identified.
  • the structures in the database are based either on experimental data, generated by ⁇ MR or x-ray crystallography, or modelled three-dimensional structures based on two-dimensional data.
  • models of compounds whose structure is in some way similar to the compound of interest are generated by a computer program using information derived from known structures and/or theoretical rules.
  • the success of both database and de novo methods for identifying compounds with activities similar to the agent of interest depends on the identification of the functionally relevant portion of the agent of interest.
  • the functionally relevant portion is referred to as a phamacophore.
  • a pharmacophore is an arrangement of structural features and functional groups important for biological activity.
  • pharmacophores may be identified which correspond to agents of the invention.
  • Such pharmacophores may allow the identification of agents with a similar structure to ILK or PSl and which may be capable of interfering with the interaction between ILK and PSl .
  • Programs suitable for generating predicated three-dimensional structures from two-dimensional data include Concord (Tripos Associates, St. Louis, MO) and 3-D Builder (Chemical Design Ltd., Oxford, UK).
  • Programs suitable for searching three-dimensional databases to identify molecules bearing a desired pharmacophore include MACCS-3D and ISIS/3D (Molecular Design Ltd., San Leandro, CA) and ChemDBS-3D (Chemical Design Ltd., Oxford, UK).
  • Programs suitable for pharmacophore selection and design include DISCO (Abbott Laboratories, Abbott Park, IL) and catalyst (Bio-CAD Corp., Mountain view, CA). Databases of chemical structures are available from Cambridge Crystallographic Data Centre (Cambridge, UK) and Chemical Abstracts Service (Columbus, OH). De novo design programs include Ludi (Biosym Technologies Inc., San Diego, CA) and Aladdin (Daylight).
  • the effect of a test agent on the activity of PKB may be determined by techniques common in the art.
  • An example of a suitable technique for determining the activity of PKB involves the isolation of PKB from a sample of interest, followed by incubation with PKB-specific substrate (Upstate) and [ ⁇ - 33 P] ATP, and subsequent detection of 33 P-labelled PKB-specific substrate (Weihl et al, 1999).
  • antibodies can be used to detect the phosphorylated form of the PKB-specific substrate peptide.
  • a peptide based around the serine 9 phosphorylation site on GSK3-beta can be used as substrate and PKB-dependent phosphorylation detected by using antibodies specific for the phosphorylated form of this peptide.
  • Phosphorylation on serine 473 can be detected by antibodies specific for the serine- 473 phosphorylated form of PKB (available commercially e.g. from New England Biolabs). Such assays could be put into a number of medium to high throughput screening technologies.
  • ⁇ -secretase activity can be tested by assessing the production of beta-amyloid by cells.
  • Gamma secretase is responsible for cleavage of beta-amyloid at position 40 or 42 (occasionally 43).
  • Antibodies specific for beta-amyloid terminating at position 40 or 42 are commonly used in e.g. an ELISA format assay (commercial kits are available for this) to measure the amount of beta-amyloid terminating at positions 40 and 42.
  • beta- amyloid terminating at position 42 is much more amyloidogenic (i.e., forms plaques more easily) than that terminating at position 40 so often the 42:40 ratio is taken as an important indicator of the likelihood of AD developing.
  • PSl transgenic mice with AD-causing mutants of PSl produce more beta-amyloid terminating at position 42 than position 40.
  • amyloid-containing tissue e.g.from post-mortem biopsy can be analysed by mass spectrometry and the amount of beta-amyloid ending at amino acid 40 and 42 determined.
  • Gamma secretase activity can also be assayed in cell membrane preparations using fusion proteins as substrate (Li et al, 2000, Nature 405:689-694).
  • test agent on apoptosis may be determined by techniques common in the art. For example, TUNEL staining performed using the Apoptosis Detection System, Fluorescein (Promega) Flow-cytometry/ FACS analysis with propidium iodide, followed by fluorescence measurement using a FACscan flow cytometer and CELLQuest acquisition and analysis software. Additional methods include a straightforward cell count and an analysis of cell morphology (cells undergoing apoptosis are morphologically distinguishable from non-apoptotic cells, e.g. under the light microscope).
  • test agent on Alzheimer's disease may be determined by techniques common in the art, e.g. by assaying amyloid deposition in the brains of mouse models.
  • modulators of the interaction between ILK and PSl do not disrupt the interaction between ILK and its other in vivo targets. It is also preferable that modulators of the interaction between ILK and PS 1 do not disrupt the interaction between PSl and its other in vivo targets. That is, modulators are generally specific for the interaction between ILK and PS 1.
  • test agents for use in methods of the invention include combinatorial libraries, defined chemical entities, peptides and peptide mimetics, oligonucleotides and natural product libraries. Test agents may be used in an initial screen of, for example, ten agents per reaction, and the agents of batches which show modulation tested individually. Furthermore, antibody products (for example, monoclonal and polyclonal antibodies, single chain antibodies, chimaeric antibodies, CDR-grafted antibodies and humanized antibodies) may be used.
  • Suitable modulators may also include functional variants of ILK or PS 1 , fragments thereof, mimetics of either ILK or PSl or a natural ligand of ILK or PSl, for example polypeptides based on ILK or PSl that mimic the structural region involved in ILK/PS 1 binding interactions, polypeptides having a sequence corresponding to a functional binding domain of either ILK for PSl or PSl for ILK and antibodies which immunoreact with either ILK, PS 1 or the ILK/PS 1 complex.
  • the region of ILK involved in binding to PSl and the region of PSl involved in binding to ILK can be determined by techniques common in the art, for example, by testing for alteration in the interaction between ILK and PSl wherein one or both of these proteins has a specified deletion in their amino acid sequence.
  • a protein which has a deletion in the region involved in ILK/PS1 binding interaction can therefore be identified by altered binding properties.
  • Modulators of the invention may be in substantially purified form. They may be in substantially isolated form, in which case they will generally comprise at least 80% e.g. at least 90, 95, 97 or 99% by weight of the dry mass in the preparation.
  • the agent is typically substantially free of other cellular components.
  • the agent may be used in such a substantially isolated, purified or free form in the method or be present in such forms in a kit.
  • the invention also provides a test kit for the identification of an agent that modulates the interaction between ILK and PSl, wherein the kit comprises: as a first component, ILK or a functional variant thereof; as a second component, PSl or a functional variant thereof; and optionally, a means for determining whether a test agent modulates the interaction between the first component and the second component.
  • a means may be the reagents and solutions required to determine whether ILK and PSl interact according to any method known in the art.
  • the invention also provides a test kit for the identification of an agent that modulates the activity of PKB or ⁇ -secretase, wherein the kit comprises: as a first component, a system comprising ILK, PSl and either PKB or ⁇ -secretase or functional variants of any of these; as a second component, an agent identifiable by a method according to any one of claims 1 to 3;and optionally a means for determining whether the second agent modulates the activity of the first component.
  • a means may be the reagents and solutions required to determine the activity of PKB or ⁇ -secretase according to any method known in the art.
  • Kits of the invention are optionally provided with packaging and preferably comprise instructions for the use of the kit.
  • Modulators of the invention may be used in a method of treatment of the human or animal body by therapy.
  • such agents may be used in a method of treatment of conditions associated with the interaction between ILK and PSl, conditions associated with the activity of PKB, conditions associated with the activity of ⁇ -secretase and conditions associated with apoptosis.
  • Such conditions include Alzheimer's disease and cancer.
  • the condition of a patient requiring a modulator of the interaction between ILK and PSl can be improved by administration of a modulator of the invention.
  • a fherapeutically effective amount of a modulator of the invention may be given to a host in need thereof.
  • Modulators of the interaction between ILK and PSl may be administered in a variety of dosage forms. Thus, they can be administered orally, for example as tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules.
  • the modulators may also be administered parenterally, either subcutaneously, intravenously, intramuscularly, intrasternally, transdermally or by infusion techniques.
  • the modulators may also be administered as suppositories.
  • a physician will be able to determine the required route of administration for each particular patient.
  • the formulation of a modulator for use in preventing or treating one of the above-mentioned conditions will depend upon factors such as the nature of the exact modulator, whether a pharmaceutical or veterinary use is intended, etc.
  • a modulator may be formulated for simultaneous, separate or sequential use.
  • a modulator is typically formulated for administration in the present invention with a pharmaceutically acceptable carrier or diluent.
  • the pharmaceutical carrier or diluent may be, for example, an isotonic solution.
  • solid oral forms may contain, together with the active compound, diluents, e.g. lactose, dextrose, saccharose, cellulose, corn starch or potato starch; lubricants, e.g. silica, talc, stearic acid, magnesium or calcium stearate, and/or polyethylene glycols; binding agents; e.g. starches, gum arabic, gelatin, methylcellulose, carboxymethylcellulose or polyvinyl pyrrolidone; disaggregating agents, e.g.
  • Such pharmaceutical preparations may be manufactured in known manner, for example, by means of mixing, granulating, tabletting, sugar-coating, or film-coating processes.
  • Liquid dispersions for oral administration may be syrups, emulsions or suspensions.
  • the syrups may contain as carriers, for example, saccharose or saccharose with glycerine and/or mannitol and/or sorbitol.
  • Suspensions and emulsions may contain as carrier, for example a natural gum, agar, sodium alginate, pectin, methylcellulose, carboxymethylcellulose, or polyvinyl alcohol.
  • the suspensions or solutions for intramuscular injections may contain, together with the active compound, a pharmaceutically acceptable carrier, e.g. sterile water, olive oil, ethyl oleate, glycols, e.g. propylene glycol, and if desired, a suitable amount of lidocaine hydrochloride.
  • Solutions for intravenous administration or infusion may contain as carrier, for example, sterile water or preferably they may be in the form of sterile, aqueous, isotonic saline solutions.
  • the invention also provides a method for the production of a pharmaceutical composition.
  • a suitable method of the invention comprises combining a pharmaceutically acceptable carrier or diluent as described above with an agent identified by a method of the invention as a modulator of the interaction between ILK and PSl, a modulator of the activity of PKB or ⁇ -secretase, or a modulator of apoptosis or Alzheimer's disease.
  • the agent used in the production of a pharmaceutical composition may be substantially isolated directly by an identification method of the invention, or may be produced de novo following its identification by a method of the invention.
  • a therapeutically effective amount of a modulator is administered to a patient.
  • the dose of a modulator may be determined according to various parameters, especially according to the substance used; the age, weight and condition of the patient to be treated; the route of administration; and the required regimen. Again, a physician will be able to determine the required route of administration and dosage for any particular patient.
  • a typical daily dose is from about 0.1 to 50 mg per kg of body weight, according to the activity of the specific modulator, the age, weight and conditions of the subject to be treated, the type and severity of the condition and the frequency and route of administration.
  • daily dosage levels are from 5 mg to 2 g.
  • Antibodies were from the following commercial sources: presenilin-1, MAB 1563 (Chemicon); FLAG antibody M2 (Sigma-Aldrich); Myc antibody 9E10 (Sigma- Aldrich). Antisera were used according to the manufacturers' directions. ILK antiserum was raised against a TrpE-ILK fusion protein encompassing the N-terminal ankyrin repeat region of ILK (residues 1-171). Fusion protein was induced and purified by preparative SDS-PAGE. Purified protein was injected into two rabbits (Eurogentec, Belgium).
  • Anti-ILK polyclonal antiserum but not pre- immune serum, specifically detected the TrpE-ILK antigen as well as both endogenous ILK and Flag-tagged ILK protein transiently expressed in COS and 293 HEK cells (data not shown).
  • ILK Clones were obtained according to Lynch et al (1999).
  • DN-ILK (ILK K->M) was constructed by PCR mutagenesis.
  • Oligonucleotides including restriction sites for BsrDl (5 prime) and (3 prime) are set forth in SEQ ID NOS: 8 and 9 respectively. These were used to amplify a 188 bp fragment containing the desired lysine to methionine change (residue 220 - in bold in SEQ ID NO: 8). This was cloned into pBluescript as an EcoRI-BamHI fragment and the change confirmed by DNA sequencing.
  • the fragment was then subcloned into pBluescript-ILK as a BsrDl -Ndel fragment (our cDNA for ILK has aNdel site at position 975 due to a silent G-A polymorphism).
  • a Notl fragment containing the ILK cDNA with the lysine to methionine mutation was subcloned into pMT2 for expression.
  • FSF motif was performed by PCR directed mutagenesis.
  • PCR primers were constructed covering and including the Ndel (SEQ ID NO: 10) and BsfXI sites.
  • the primer covering the BstXI site also included the following amino acid substitutions FSF to FEF, as set forth in SEQ ID NO: 11.
  • the fragment was amplified by PCR using Pfu DNA polymerase (Stratagene) and subcloned into pMT2-ILK as an Ndel-BstXI fragment.
  • Pfu DNA polymerase (Stratagene)
  • subcloned into pMT2-ILK as an Ndel-BstXI fragment.
  • N-Flag ILK and C-Flag ILK a Flag tag was added to either the N- or C- terminus of ILK by PCR mutagenesis and the insert subcloned into the pMT2 vector.
  • the ILK sequences in all plasmids were then verified by DNA sequencing. Presen
  • the cDNA for PS-1 was cloned into the vector pCDNA3 (invitrogen). Mutants M146N, C263R and delta 9 were constructed by oligo-directed mutagenesis and sub-cloned into the same vector.
  • EYFP enhanced yellow fluorescent protein - Clontech
  • the coding sequences for dishevelled 1 and dishevelled 2 were inserted into the expression vector pCD ⁇ A-3 (Invitrogen).
  • the p85 alpha subunit of PI3-kinase was obtained as described in Otsu et al, 1991, Cell 65:91-104.
  • COSl cells were grown in Glasgow's modified Eagles medium (Life Technologies) supplemented with 10% v/v newborn calf serum and 2mM L- glutamine. COSl cells were transfected in 6 well plates using Fugene 6 (Boehringer) according to the manufacturers instructions and left for 72h before harvest. Prior to harvest the cells were serum starved for 16h. For harvesting, cells were washed twice in ice cold phosphate buffered saline (PBS) and lysed for 15min on ice in radioimmune precipitation assay (RIPA) buffer.
  • PBS ice cold phosphate buffered saline
  • RIPA radioimmune precipitation assay
  • RIPA buffer 0.5% w/v sodium deoxycholate, 150mM sodium chloride, 1% v/v P40, 50mM Tris-HCL pH 8.0, 0.1% w/v SDS, 10% v/v glycerol, 5mM EDTA, ImM sodium orthovanadate, ImM PMSF, lOmg ml "1 aprotinin, lOmg ml "1 leupeptin. Insoluble material was removed by centrifugation (12000g for lOmin at 4C).
  • Cells were lysed in ILK lysis (buffer 150 mM ⁇ aCl, l%(v/v) ⁇ P-40, 0.5% (w/v) sodium deoxycholate, 50 mM HEPES pH 7.5, 1 microgram ml "1 each leupeptin and aprotinin, 50 microgram ml "1 phenylmethylsulphonyl fluoride. 0.25-lmg of total protein lysate was immunoprecipitated, with lmg ml "1 of appropriate antibody for 1- 24h at 4C.
  • COS cells were transiently co-transfected with a plasmid bearing the wild type (wt) ILK coding sequence, together with either wt PS-1, or PS-1 mutants associated with early onset FAD (PS-1 M146N, PS-1 C263R).
  • Western blot analysis of whole cell ly sates with affinity purified polyclonal anti-ILK antibodies revealed a 59 kDa protein, corresponding to exogenously expressed ILK, in transfected cells (Figure 1A).
  • Low level expression of endogenous ILK protein in mock-transfected cos cells was also detected after longer exposure.
  • ILK mutants which we have shown induce differential activation of PKB (ILK FEF, ILK KM, ILK kinase domain alone), for their ability to associate with PS-1. All ILK mutants tested showed approximately equal association with PS-1 compared with wtlLK.
  • ILK FEF ILK FEF
  • S-»E point mutation within a candidate phosphorylation site within the region of ILK corresponding to the catalytic loop
  • EYFP-tagged PS-1 was transiently transfected into COS cells, together with a plasmid vector bearing either wt ILK, or the p85alpha subunit of PI3-kinase, or the empty vector alone.
  • Western blot analysis of whole cell lysates using anti-p85alpha-specific antibodies confirmed high level expression of p85alpha in transfected.
  • ILK immunoprecipitates contained abundant amounts of complexed PS-1 protein ( Figure 4B lane 3)
  • no PS-1 protein was recovered from anti-p85 alpha immunoprecipitates ( Figure 4B lane 6).

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Abstract

Méthode d'identification d'un agent modulant l'interaction entre une kinase liée à l'intégrine (ILK) et la préséniline-1 (PS1), caractérisée (a) en ce qu'on met en oeuvre, comme premier composant, ILK ou un variant fonctionnel de celui-ci ; (b) on met en oeuvre, comme second composant, PS1 ou un variant fonctionnel de celui-ci ; (c) on met en contact le premier et le second composants en présence d'un agent d'essai dans des conditions qui, en l'absence de cet agent d'essai, permettraient aux deux composants de réagir mutuellement ; et (d) en ce qu'on détermine si l'agent d'essai est capable de moduler l'interaction entre le premier et le second composants, de façon à déterminer si l'agent d'essai est un modulateur de l'interaction entre ILK et PSI. Un tel agent peut être utilisé dans le traitement de conditions associées à une apoptose ou à la maladie d'Alzheimer.
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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005083436A1 (fr) * 2004-02-26 2005-09-09 Bayer Healthcare Ag Methodes diagnostiques et therapeutiques destinees au traitement de maladies associees a la kinase 1 liee a l'integrine (ilk1)
WO2005083437A1 (fr) * 2004-02-26 2005-09-09 Bayer Healthcare Ag Methodes diagnostiques et therapeutiques destinees au traitement de maladies associees a la kinase 2 liee a l'integrine (ilk2)
US7750037B2 (en) 2002-03-01 2010-07-06 Takeda Pharmaceutical Company Limited Antidepressant

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* Cited by examiner, † Cited by third party
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CA2239151C (fr) * 1995-12-21 2011-03-29 Shoukat Dedhar Kinase liee a une integrine, inhibiteurs de cette kinase, traitement medical faisant appel a ces inhibiteurs, therapie genique et inhibiteurs du type pseudo-substrats
EP0876483A1 (fr) * 1996-01-26 1998-11-11 HSC Research and Development Limited Partnership Acides nucleiques et proteines lies a la maladie d'alzheimer et leurs utilisations

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7750037B2 (en) 2002-03-01 2010-07-06 Takeda Pharmaceutical Company Limited Antidepressant
WO2005083436A1 (fr) * 2004-02-26 2005-09-09 Bayer Healthcare Ag Methodes diagnostiques et therapeutiques destinees au traitement de maladies associees a la kinase 1 liee a l'integrine (ilk1)
WO2005083437A1 (fr) * 2004-02-26 2005-09-09 Bayer Healthcare Ag Methodes diagnostiques et therapeutiques destinees au traitement de maladies associees a la kinase 2 liee a l'integrine (ilk2)

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