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WO2008135790A1 - Fragment c-terminal biologiquement actif de l'acétylcholinestérase - Google Patents

Fragment c-terminal biologiquement actif de l'acétylcholinestérase Download PDF

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WO2008135790A1
WO2008135790A1 PCT/GB2008/050326 GB2008050326W WO2008135790A1 WO 2008135790 A1 WO2008135790 A1 WO 2008135790A1 GB 2008050326 W GB2008050326 W GB 2008050326W WO 2008135790 A1 WO2008135790 A1 WO 2008135790A1
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peptide
ache
agent
disease
nachr
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Susan Adele Greenfield
Cherie E. Bond
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ENKEPHALA Ltd
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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)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/18—Carboxylic ester hydrolases (3.1.1)
    • 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
    • C12Q1/44—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving esterase
    • C12Q1/46—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving esterase involving cholinesterase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y301/00—Hydrolases acting on ester bonds (3.1)
    • C12Y301/01—Carboxylic ester hydrolases (3.1.1)
    • C12Y301/01007—Acetylcholinesterase (3.1.1.7)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G01N33/6896—Neurological disorders, e.g. Alzheimer's disease
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to the isolated polypeptide consisting of the C-terminal 30 amino acid residues of the T-isoform of the enzyme acetylcholinesterase (T-AChE).
  • This polypeptide fragment designated T30 peptide, exhibits non-enzymatic biological activities consistent with a role in causation of neurodegenerative disease. Hence, it is of interest in relation to both diagnosing, and providing new treatments for, such disease, especially Alzheimer's Disease, Parkinson's Disease and Motor Neuron Disease.
  • AD Alzheimer's disease
  • PD Parkinson's disease
  • MND Motor Neuron disease
  • AD Alzheimer's disease
  • PD Parkinson's disease
  • MND Motor Neuron disease
  • One familiar idea is that, for AD at least, the key pathological process is the formation of amyloid plaques and fibrils, derived from abnormal cleavage of amyloid precursor protein (APP).
  • APP amyloid precursor protein
  • the 'Amyloid Hypothesis' Hardy & Higgns, Science (1992) 256, 84-85, 'Alzheimer's disease: the amyloid cascade hypothesis'
  • AD Alzheimer's disease
  • PD Parkinson's disease
  • MND Motor Neuron disease
  • AD acetylcholine
  • AChE can give rise to associated non-enzymatic action in the brain and that this could provide an underlying causation for neurodegenerative disease
  • all isoforms of AChE are derived from a single gene transcript and contain the invariable exons 2, 3 and 4.
  • the T-AChE isoform arises through alternative imRNA splicing of exon 6 to the invariable exons.
  • T-AChE upregulation correlates with early developmental events and stress responses
  • evidence suggests that the T-AChE isoform is associated with neurodegenerative processes (Sternfeld et al. (2000) Proc. Natl Acad, Sci. USA 97, 8647-8652; Zhang et al. (2002) Cell Death Differen. 9, 790-800).
  • T- AChE reverts to a more immature developmental form (G1 ) (Arendt et al. (1992) Neurochem.lnt.
  • AChE amyloid beta- peptide
  • AD Alzheimer & Cuello (1984) Lancet 1_, 513; Shortridge et al. (1985) Clin. Neuropathol. 4,227-237; Greenfield 1996 ibid.
  • AChE and amyloid beta- peptide (A ⁇ P) have been co-localized to senile plaques in AD (Inestrosa et al. (2005) Subcell. Biochem. 38, 299-317) and AChE increases the neurotoxicity of amyloid fibrils (Inestrosa et al. (1996) Neuron 16 , 881-891 ; Alvarez et al. (1998) J.
  • AChE and the amyloid precursor protein (APP) exhibit functional similarities and co-dependence. Both proteins are transiently expressed during specific developmental stages, display properties of cell adhesion molecules and are secreted by neurons and glia. AChE and APP are decreased in cerebrospinal fluid (CSF) of AD patients (Appleyard et al. (1983) Lancet U_, 452; Arendt et al. (1984) Lancet 1_, 173; Farlow et al. (1992) Lancet 340, 453-454) and both proteins can enhance calcium entry into cells (Webb et al. (1996) Eur. J. Neurosci.8, 837-841 ; Ueda et al.
  • CSF cerebrospinal fluid
  • AChE can induce the expression of APP and accumulation of A ⁇ P (von Bernhardi et al. (2003) Neurobiol. Dis. J_4, 447-457) and, conversely, A ⁇ P induces AChE production (Saez-Valero et al. (2003) Biochem. 43, 15292-15299) in both neurons and glia, suggesting that the synthesis and metabolism of these molecules are linked physiologically.
  • AChE plays an integral role in neurodegenerative pathology.
  • the C-terminal region of the T-AChE protein has many structural and functional similarities to the N-terminus of the predominant amyloid beta protein (A ⁇ 42) found in
  • AD The 14 amino acid residue portion corresponding to Synaptica Peptide, within the
  • T30 peptide has high homology to the N-terminal region of A ⁇ 42 (Greenfield & Vaux
  • Figure 2 shows the sequence alignment of the amyloid precursor protein and T-AChE with the relevant peptides (T30, Synaptica Peptide (T14) and A ⁇ 42) highlighted for comparison.
  • T-AChE and R-AChE have identical sequences except for their C-terminal exons (see Figure 3). Moreover, it has previously been reported that a natural C-terminal truncated form of T-AChE is present in fetal bovine serum relying on sequencing of tryptic peptides (Saxena et al. (2003) Biochem. 43, 15292-15299, 'Natural Monomeric Form of Fetal Bovine Acetylcholinesterase Lacks the C-terminal Tetramerization Domain'). However, the truncated monomer size given is 543-547 compared to 583 for mature T- AChE and no information was provided by Saxena et al. on any C-terminal fragment, or more especially reason to isolate and investigate properties of the C terminal 30 amino acid residues of T-AChE.
  • WO 00/73427 suggests use of the C-terminal region of R-AChE or T-AChE as a source of therapeutic peptides, although the data provided, if anything, makes a case for focussing on the C-terminal 26 mer of R-AChE (ARP) and only the C-terminal 40 mer of T-AChE was tested. There is no suggestion of any isoform of AChE being a source of any peptide having physiological relevance to disease causation; WO 00/73427 provides no indication that unusual properties beyond those investigated might be found by selecting the C-terminal 30 amino acid residues of T-AChE. Thus, it is to be noted that only the much shorter T14 peptide was previously recognised as exhibiting properties consistent with a mechanism for neurodegeneration
  • T30 peptide has been shown in live cell binding experiments to bind like Synaptica Peptide to alpha 7 nicotinic acetylcholine receptors, but to bind with higher affinity to the allosteric modulation site targeted by T14 and to have a second binding site on the receptor.
  • ⁇ 7-nicotinic acetylcholine receptor ( ⁇ 7-nAChR) is a potent calcium ionophore, which has been suggested to be pivotal in neural development, neural responses to injury and neurodegeneration. Consistent with its role in degenerative processes, ⁇ 7- nAChR mRNA is upregulated in affected areas of the AD brain (Nagele et al. (2002) Neurosci. 110, 199-211 ) and in transgenic mice overexpressing APP. A ⁇ 42 and T14 peptide exhibit modulatory activity at the ⁇ 7-nAChR and potentiate calcium influx through L-type voltage gated calcium channels (L-VGCC) (Dineley et al.
  • L-VGCC L-type voltage gated calcium channels
  • a ⁇ P binds ⁇ 7-nAChR with picomolar affinity (Wang et al. (2000) J. Neurochem.75, 1 155-1161 ), promoting rapid Ca 2+ influx into hippocampal neurons and activating kinase signalling cascades. Moreover, during critical developmental periods, the transient expression of this receptor correlates spatially and temporally with the transient appearance of AChE (Taylor et al (1994) Biochem. Soc. Trans. 22, 740-745; Broide et al. (1996) J. Neurosci. 16, 2956-2971 ).
  • T30 peptide binds to the ⁇ 7-nAChR, coupled with the fact that it can be generated by a single protease cleavage in vivo (see in Figure 4 the indicated beta-secretase cleavage site at the N-terminus of the T30 sequence), means that it is now proposed as a physiologically relevant peptide of particular interest in relation to, for example, screening for agents of potential use in treating neurodegenerative disease, especially for example AD, PD and MND. It is postulated that such agents also include agents which inhibit proteolytic release of the T30 peptide from T-AChE.
  • the findings now presented also open new possibility for diagnosis of neurodegenerative disease, for example AD, PD and MND, especially AD.
  • T30 peptide As an isolated peptide having particularly significant properties for use as a tool in advancing diagnosis and treatment of neurodegenerative disease.
  • the present invention therefore provides an isolated polypeptide (T30 peptide) consisting of the C-terminal 30 amino acid residues of the T isoform of acetylcholinesterase (T-AChE), said polypeptide consisting of the sequence
  • Such functional analogues will be understood to retain substantially the same biological activity as T30 peptide in any of the tests for biological activity set out in the examples and like T30 peptide itself to be distinguishable from T14 peptide (AEFH RWSSYMVHWK; SEQ. ID no.2) in such tests.
  • T30 peptide may be selected on the basis of binding ability to the ⁇ 7-nAChR and will show higher affinity binding to the modulatory site targeted by T14 than T14 itself and binding to the same second site as T30.
  • the T30 peptide binds the ⁇ 7-nAChR with picomolar affinity whereas T14 is less potent at this receptor.
  • the data presented in Example 1 below is consistent with T30 interacting with both the active ligand binding site and the allosteric modulatory site on the ⁇ 7-nAChR.
  • isolated will equate with isolation in soluble form, although a polypeptide of the invention may for some purposes be immobilized on a solid support.
  • a polypeptide of the invention may be provided as part of a fusion construct. It may also be labelled.
  • the invention provides use of a polypeptide of the invention as an antigen and/or screening agent to provide an antibody or antibody fragment which binds T30 peptide.
  • Antibodies or antibody fragments thus obtained may be of value, for example, for use in detecting the T30 peptide for diagnostic purpose.
  • the invention provides a method of diagnosing in an individual a neurodgenerative disorder, especially AD, PD or MND, most especially AD, which comprises detecting an abnormal T30 peptide level in a cerebrospinal fluid (CSF) sample.
  • a neurodgenerative disorder especially AD, PD or MND, most especially AD
  • CSF cerebrospinal fluid
  • Such a polypeptide as a screening tool to screen for agents capable of antagonising biological activity of the polypeptide in one or more in vitro tests for such activity.
  • agents may be of interest as therapeutic agents for treatment of neurodegenerative disorders, possibly after modification or packaging for delivery across the blood-brain barrier.
  • an agent which prevents proteolytic cleavage of T30 peptide from T-AChE in the brain for use in treating a neurodegenerative disorder, for example AD, PD or MND, especially, for example, AD.
  • agents may include antisense oligonucleotides, antisense PNAs and siRNAs, which target expression of, for example, the brain ⁇ -secretase implicated in production of T30 peptide in vivo (see again Figure 4). Again such agents may be modified or packaged for delivery across the blood-brain barrier.
  • the invention also extends to pharmaceutical compositions containing such an agent together with a pharmaceutical carrier or diluent.
  • T-AChE is exposed to physiological proteolytic conditions whereby it is cleaved to release T30 peptide and such assays form a still further aspect of the invention.
  • FIG. 1 Summary of the major protein isoforms of derived form the AChE gene.
  • E- AChE is specifically found in blood cells, whereas R-AChE and T-AChe, as already noted above, are the predominant isoforms in brain tissue.
  • T-AChE is the adult CNS isoform that can be post-translationally modified to allow formation of membrane-bound tetramers, whereas R-AChE always remains monomeric, soluble and secretable.
  • Globular forms represent the monomeric and multimeric assemblies of individual subunits.
  • FIG. 2 Sequence alignment of the amyloid precursor protein (APP) and the C- terminal region of T-AChE with the T30 peptide of AChE and A ⁇ 42 fragment of APP light shaded. Also shown by dark shading are the T14 (Synaptica Peptide) portion of the T30 peptide and the homologous portion of A ⁇ 42.
  • APP amyloid precursor protein
  • FIG. 3 Schematic diagram showing the C-terminal exons of R-AChE and T-AChE and the AChE and control polypeptides used in the examples.
  • all isoforms of AChE are derived from a single gene transcript and contain the invariable exons 2, 3 and 4.
  • the T-AChE isoform arises through alternative mRNA splicing of exon 6 to the invariable exons.
  • Truncated AChE (T548) is a recombinant protein, translated from cDNA containing exons 2, 3, and 4, but lacking a C-terminal exon, which was produced from the glycophospholipid-linked form of mouse AChE (Marchot et al.
  • Figure 4 Diagram showing protease cleavage sites in APP and the C-terminal region of T-AChE. Cleavage sites for ⁇ - and ⁇ -secretase are indicated. Small arrows indicate sites cleaved in APP by other proteases. Lightly shaded box highlights A ⁇ 42. Dark shaded boxes indicate area of homology between APP and the Synaptica Peptide (T14 region) of T-AChE.
  • FIG. 5 Results of binding of T30 and T14 peptides to the ⁇ 7-nAChR in live cell binding assays.
  • MLA methylyllcaconitine
  • ⁇ BTX ⁇ - bungarotoxin
  • Ach acetylcholine
  • IVM invermectin
  • B Displacement of [ 125 I]-O- bungarotoxin with T14 and T30 peptides at a various concentrations.
  • Figure 6 Western blot analysis showing upregulation of ⁇ 7nAChR expression in cultured GH4-h ⁇ 7 cells expressing the receptor following treatment with T30 and T14 peptides. Representative western blots of ⁇ 7-nAChR protein levels in control (C) and peptide (T14, T30) treated GH4-h ⁇ 7 cells. All experiments were performed a minimum of 2-3 times.
  • C control
  • T14, T30 peptide
  • All experiments were performed a minimum of 2-3 times.
  • A Protein levels as assessed in total cell homogenate and in membrane compartments after 6 or 24 hr peptide exposure. The filled and open arrow heads indicate ⁇ 7-nAChR and actin at 55 and 42 kDa MW, respectively. Actin was used as an internal standard.
  • B Protein levels as assessed in total cell homogenate and in membrane compartments after 6 or 24 hr peptide exposure. The filled and open arrow heads indicate ⁇ 7-nAChR and actin at 55 and 42 k
  • Figure 7 Results showing increased astroglia cell proliferation/metabolism in the presence of low concentrations of T30 and T14 peptides by assay determining bioreduction by metabolically active cells, of a tetrazolium compound (3-(4,5- dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt; MTS) to form a coloured formazan product.
  • a tetrazolium compound 3-(4,5- dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt; MTS
  • Figure 8 Determination of secreted AChE, secreted BuCHe and intracellular AChE in astroglia cell culture when such cells are exposed to T30 and T14 peptides for 24 hrs.
  • Figure 9 Effect of T30 and T14 peptides on substrate inhibition of AChE.
  • Figure 10 RT-PCR analysis of ⁇ 7-nAChR mRNA showing upregulation of ⁇ 7-nAChR mRNA in GH4-h ⁇ 7 cells after 24 hrs exposure to T30 and T14 peptides. GAPDH expression was used as an internal standard. Data shown are representative results of experiments performed a minimum of 3 times.
  • A. Effect of varying concentrations of AChE peptides T14 and T30 on ⁇ 7-nAChR expression.
  • B Effect of varying concentrations of AChE peptides T14 and T30 on ⁇ 7-nAChR expression.
  • Figure 1 1 Schematic diagram showing envisaged role of APP and AChE in neurodgeneration.
  • Figure 12 Acute live cell binding in GH4-h ⁇ 7 cells.
  • Cells were pre-treated with peptides or drugs for 30 min, then [ 125 I] ⁇ -bungarotoxin ( ⁇ -BTX) was added and cells were incubated for 1.5 hr at 37 0 C.
  • Data shown is the average ⁇ SEM of 3 separate experiments.
  • A. Raw saturation binding data shows total, nonspecific, and specific binding for [ 125 I] ⁇ -BTX to ⁇ 7-nAChR in this cell line.
  • B Specific binding of [ 125 I] ⁇ -BTX to ⁇ 7-nAChR in fmol/mg protein with Scatchard analysis.
  • C C.
  • FIG. 13 Acute membrane binding in GH4-h ⁇ 7 cells. Purified membranes (50-100 ⁇ g) from GH4-h ⁇ 7 cells were incubated with peptides and/or drugs, 2 nM [ 125 I] ⁇ -BTX, and binding buffer overnight at 4 0 C. Data shown are the combined results of a minimum of 2 experiments each performed in triplicate and expressed as percent control specific binding.
  • B Effect of varying concentrations of T30 on ⁇ -BTX competition binding with ACh, MLA, and choline.
  • C Competition binding curve for ACh vs. ACh + T30 (100 nM).
  • D Competition binding curve for MLA vs.
  • T30 peptide of the invention and functional analogues thereof may be produced synthetically or by recombinant methods well known in the peptide synthesis field. In identifying such functional analogues, or antagonists of T30 peptide biological activity, one or more of the following identified biological effects of T30 peptide may be relied upon:
  • tests for such biological effects may, for example, conform with the assay systems described in the examples. It is notable that in the case of all the biological effects so determined using the T30 and T14 peptides, T30 peptide was more potent or more efficacious than the T14 peptide.
  • Functional analogues of T30 peptide thus identified may include variants having one or more additions and /or deletions and /or substitutions, e.g. one or more conservative substitutions, compared to SEQ. ID No. 1 and which exhibit substantially the same activity or activities as T30 peptide tested. As indicated above, in all instances distinction will be maintained compared to activity of T14 peptide in the same test.
  • T30 peptide As potential therapeutics for use in treating neurodegenerative disease, it may be particularly preferred to look at the ability of test agents to inhibit binding of T30 peptide to the ⁇ 7-nAChR.
  • membranes or cultured cells may be employed presenting functional ⁇ 7-nAChR or a functional analogue thereof.
  • Such a receptor will exhibit in the presence of Ca 2+ ions and acetylcholine induced Ca 2+ ion flux which can be blocked by ⁇ -bungarotoxin and modulated by T30 peptide binding at the allosteric modulation site, also targeted by T14.
  • Such a receptor may be a native homomeric ⁇ 7-nAChR, preferably a human ⁇ 7- nAChR, in its normal membrane environment.
  • synthetic membranes, or more preferably cultured cells transformed to express a functional recombinant ⁇ 7-nAChR may be employed, e.g. Xenpous oocytes or other cells engineered to express human ⁇ 7-nAChR as described in Published International Applications nos WO 94/20617 and WO 01/73446 and by Seguela et al. (J. Neurosci. (1993) 13, 596-604).
  • SH-EP1-h ⁇ 7 cells or GH4-h ⁇ 7 cells may, for example, be preferred, both of which express high levels of AChE and the human ⁇ 7-nAChR.
  • SH- EP1-h ⁇ 7 cells are human epithelial— like clonal cells derived from the human SK-N-SK human cell line that have been stably transfected to express human ⁇ 7-nAChR.
  • GH4- h ⁇ 7 cells are a well-known rat pituitary tumour-derived cell line, again transfected to express the human ⁇ 7-nAChR.
  • binding of T30 peptide alone or in the presence of the test agent may be determined as in Example 1.
  • a potential therapeutic identified as an antagonist as above may be formulated into a pharmaceutical composition together with pharmaceutical carrier or diluent. Where delivery is desired across the blood-brain barrier, as indicated above it may be desirable to modify or package the antagonist to facilitate such delivery.
  • an isolated polypeptide of the invention may be used to as antigen and/or a screening agent to obtain antibodies or antibody fragments which bind T30 peptide, including such monoclonal antibodies and fragments thereof. Production of such antibodies may be followed by labelling with detectable label for use in assays.
  • assays may include diagnostic assays as noted above.
  • diagnostic assays for neurodegenerative disease relying on identification of abnormal T30 peptide level in CSF fluid samples may take many formats, the critical requirement being to distinguish T30 peptide from AChE and other peptides/proteins sharing homology with T30 peptide in the sample.
  • anti-T30 antibodies may be employed together with other peptide/protein separation techniques well known in the diagnostic assay field.
  • Such a diagnostic assay may be combined with other clinical observations in making diagnosis of a particular neurodegenerative disorder such as Alzheimer's Disease or Parkinson's Disease.
  • T30 peptide action in the brain e.g. by inhibiting binding of T30 peptide to ⁇ 7-nicotinic receptors
  • another immediately evident approach to treating neurodegenerative disease is to intervene with processing of T-AChE to produce T30 peptide.
  • processing is consistent with action of ⁇ -secretase on AChE and hence, agents which target ⁇ -secretase including for example, antisense oligonucleotides or PNAs or siRNAs which target expression of that protease are of immediate interest in this connection.
  • agents which target ⁇ -secretase including for example, antisense oligonucleotides or PNAs or siRNAs which target expression of that protease are of immediate interest in this connection.
  • Such an agent may be an antibody or antibody fragment.
  • other agents which will inhibit processing of AChE to give the T30 peptide may be screened for.
  • Such a screen may take the form of a specific assay for ⁇ -secretas
  • the invention moreover provides a method of identifying a potential therapeutic agent for use in treating a neurodegenerative disease which comprises contacting a test agent with T-AChE exposed in vitro to physiological proteolytic conditions whereby in the absence of the test agent it is cleaved to release T30 peptide and determining whether such cleavage is prevented.
  • Means for packaging or modifying nucleic acids and polypeptides for delivery across the blood-brain barrier are known and may possibly be employed for delivering therapeutic agents as discussed above into the brain, but direct delivery into the brain may also be considered. It will be understood that treatment may include prophylactic treatment or treatment at an early stage before symptoms of neurodegeneration are observable.
  • Astroglia were prepared as previously described (Whyte & Greenfield (2003) Exp. Neurol. 184, 496-509: ' Effects of acetylcholinesterase and butrylcholinesterase on cell survival, neurite outgrowth and voltage-dependent calcium currents of embryonic ventral mesencephalic neurons'). Briefly, P1-P3 Wistar rats were treated with an overdose of isofluorane anaesthetic (Schedule 1 , Animal Scientific Procedures Act, UK, 1986), and then decapitated in a sterile environment.
  • the cerebrum was removed by blunt dissection, rolled on sterile filter paper to remove meninges, cut into ⁇ 1 mm 3 pieces, and dissociated with gentle trituration in Dulbecco's modified Eagle's medium with 4500 mg/l glucose and GlutaMAX (DMEM; Life Technologies Ltd., Paisley, UK) containing 10% fetal calf serum, 1 % penicillin/streptomycin, and 2.5 ⁇ g/ml amphotericin B.
  • the dissociated tissue was plated into 75 cm 2 flasks precoated with poly-D-lysine (PDL), then incubated at 37 ° C in a humidified atmosphere (95% air: 5% CO 2 ) for 7 days.
  • GH4-h ⁇ 7 cells (Merck & Co, Inc, Rahway, USA) were maintained in DMEM (4500 mg/l glucose with GlutaMAX containing 10% fetal bovine serum, 1 % penicillin/streptomycin, 2.5 ⁇ g/ml amphotericin B and 500 ⁇ g/ml active G418 (geneticin).
  • Ca 2+ -free, serum-free media was prepared with calcium-free DMEM (4500 mg/ml glucose; Invitrogen, Paisley, UK), 25 mM HEPES, 2 mM GlutaMAX, 1 % penicillin/streptomycin, 2.5 ⁇ g/ml amphotericin B, and 1.8 mM BaCI 2 .
  • Medium was sterilized by filtration through a 0.22 ⁇ m polyethersulfone low protein binding membrane and stored at 4 ° C.
  • Confluent cells were seeded into 12-well plates at a density of 5 x 10 4 cells/well and allowed to recover for 24-48 hours before experimentation. Live cell binding was performed by treating cells with indicated peptides or ⁇ 7-nAChR inhibitors for 1 hr at 37 ° C in cell media with 1 % FBS. Then [125l]- ⁇ -bungarotoxin (150 Ci/mmol, GE Healthcare Bio-Sciences, Amersham, UK) was added and cells were incubated at 37 ° C for a further 1.5 hr. Cell layers were washed 3x with 2 ml cell media, then 0.5 ml 1 M NaOH was added to each well to lyse cells. Cell lysates were transferred to 5 ml scintillation fluid and radioactivity was determined using a Beckman LS6000IC scintillation counter.
  • the supernatants were combined and centrifuged at 50,000 rpm (70 Ti rotor) for 30 min (Beckman Ultracentrifuge).
  • the membrane pellet was washed with 5 ml lysis buffer and centrifuged as above. All centrifugations were carried out at 4 ° C.
  • the pelleted membranes were resuspended in binding buffer (50 mM Tris-HCI, 120 mM NaCI, 5 mM KCI, 1 mM MgCI 2 , 2.5 mM CaCI 2 , pH 7.0) and protein concentration determined using the DC Protein Assay kit (Bio-Rad Laboratories, Ltd., Hemel Hempstead, UK).
  • Binding assays were assembled on ice in borosilicate glass test tubes with 50-100 ⁇ g membrane protein in binding buffer in a final volume of 250 ⁇ l. Binding reactions were incubated at 4 ° C overnight, and then terminated by rapid vacuum filtration using a Brandel Cell Harvester onto Whatman GF/B glass fibre filters pre-soaked in 0.4% polyethylenimine.
  • Confluent cells were seeded into 96-well plates at a density of 1x10 4 cells/well and allowed to recover for 24 hr before experimentation. Cells were treated with peptides at indicated concentrations for 48 hours, then cell proliferation was measured using the CellTiter 96 Aqueous One Solution Cell Proliferation Assay Kit (Promega, Southampton, UK) as per the manufacturer's instructions.
  • This assay is based on the bioreduction, by metabolically active cells, of a tetrazolium compound (3-(4,5- dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt; MTS) to form a colored formazan product that is soluble in tissue culture medium.
  • a tetrazolium compound 3-(4,5- dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt; MTS
  • 100 ng cDNA was amplified by PCR with 50 pmol gene-specific primers, 1.5 mM MgCb, 200 ⁇ M dNTPs, and 1.25 U Taq DNA polymerase (Promega, Southampton, UK) in a 50 ⁇ l final reaction volume. After an initial denaturation of 95 ° C for 2 min, reactions were amplified for 35 cycles: 95 ° C for 30 sec, 55 ° C for 30 sec, 72 ° C for 1 min, followed by a final extension of 72 ° C for 10 min. Reaction products were separated by electrophoresis on 1.5-2.0% agarose TAE gels and visualized by UV illumination. Images were captured and band density determined using a Bio-Rad Gel Doc 2000 and QuantityOne software (Bio-Rad, Hempstead, UK).
  • Cells were harvested in Tris HCI 25 mM containing EDTA 2 mM and a complete set of protease inhibitors (Complete; Roche, Mannheim, Germany) and pelleted at 4 0 C and 10,000 x g for 10 minutes.
  • lysis buffer containing Tris HCI 25 mM pH 7.4, NaCI 150 mM, EDTA 2 mM, phenylmethylsulfonyl fluoride PMSF 0.1 mM, Nonidet 0.1 % and a complete set of protease inhibitors was added. Cells were vortexed vigorously for 5 minutes placing on ice intermittently.
  • cell lysates were pelleted by centrifugation for 50 minutes at 4 0 C and 100,000 x g. The resulting pellets (membranes) were resuspended in lysis buffer and protein levels (alpha7 nAChR and actin) were determined by Western blot analysis. To this end, protein amount in both total homogenates and membrane lysates was determined using the method established by Bradford (Bradford (1976) Anal. Biochem. 72, 248-254).
  • Example 1 Binding of the T30 and T14 peptides to the ⁇ 7-nicotinic acetylcholine receptor ( ⁇ 7nAChR).
  • Binding of the T30 and T14 peptides to the ⁇ 7nAChR was investigated employing live cells expressing the receptor (GH4-h ⁇ 7 cells) in culture as described above.
  • T30 binds the ⁇ 7-nicotinic acetylcholine receptor ( ⁇ 7nAChR).
  • ⁇ 7nAChR ⁇ 7-nicotinic acetylcholine receptor
  • T30 shows higher affinity than T14 for their common binding site and has an additional effect at a second binding site on the receptor (see Figure 5).
  • T14 exhibited approximately 40% efficacy at 10 ⁇ M concentration, whereas the same concentration of T30 was 70% efficacious. Higher concentrations of these peptides did not further displace radioligand binding. In contrast, none of the control peptides were able to compete with ⁇ -BTX for binding to the ⁇ 7-nAChR (Fig. 12D). Similarly, neither full-length T-AChE, nor truncated T548, had an effect on ⁇ -BTX binding to the receptor (Fig. 12D).
  • Ligand IC50 t SEM Ki ⁇ ⁇ SEM
  • Table 1 Comparison of EC50 and K 1 values showing the effect of increasing concentrations of T30 on choline binding to the ⁇ 7-nAChR.
  • Example 2 Effect of the T30 and T14 peptides on expression of the ⁇ 7-nAChR in GH4- h ⁇ 7 cells
  • GH4-h ⁇ 7 cells were treated for 6 hours or 24 or more hours with 100 nM of T14 or T30 peptide.
  • Preliminary Results ⁇ 7-nAChR protein expression increases after chronic treatment (24-48 hr) with T14 or T30 peptide. This increased expression has been specifically localized to the plasma membrane indicating that the peptides stimulate increased production or trafficking of receptors to the cell surface. T30 induces significantly more protein expression than than does T14 (see Figure 6).
  • Table 2 Summary of saturation binding parameters showing the effects of chronic T- AChE peptide treatment on the number of ⁇ 7-nAChR binding sites (B max ) and receptor affinity (K d ) for ⁇ -BTX.
  • Example 3 Effect of T30 and T14 peptides on proliferation/metabolism of astrogial cells as measured by MTS assay
  • T14 and T30 exhibit equal cytotoxic effects at high concentrations (-20% inhibition of cell proliferation at 10 ⁇ M)
  • the proliferative effect of low concentrations on astroglia is greater with T30 (120 ⁇ 4% at 1 pM and 135 ⁇ 9% at 10 pM) than with T14 (96 ⁇ 5% at 1 pM and 121 ⁇ 8% at 10 pM); see Figure 7.
  • Example 4 Effect of T30 and T14 peptides on astroglial cell AChE intracellular activity and secretion of cholinesterases
  • Substrate inhibition is a characteristic of AChE activity whereby excessive concentrations of substrate cause a feedback inhibition of the enzymatic activity.
  • T30 and T14 decrease substrate inhibition thus enhancing AChE activity at higher concentrations of substrate.
  • T30 demonstrates a significantly greater effect than does T14 (see Figure 9).
  • Example 6 Effect of T30 and T14 peptides on gene expression in GH4-h ⁇ 7 cells
  • RT minus controls were negative and gene expression in control cells did not change noticeably throughout the series of experiments.
  • RT-PCR analysis was performed in control GH4-h ⁇ 7 cells and those exposed to AChE peptides at concentrations ranging from 1 nM to 1 ⁇ M for 24 hr (Fig. 10A).
  • T14 or T30 peptide treatment ⁇ 7-nAChR mRNA expression was markedly upregulated for all concentrations of the peptides tested.
  • Levels of ⁇ 7-nAChR mRNA displayed a concentration-dependent increase with T14 treatment, with maximal expression at 100 nM.
  • a similar high level of ⁇ 7-nAChR expression was achieved after treatment with only 1 nM T30.
  • ⁇ 7-nAChR expression levels declined, however they remained significantly enhanced as compared with controls at all peptide concentrations tested.
  • peptide-treated GH4-h ⁇ 7 cells were exposed to the cross-linking agent BS 3 prior to harvesting for analysis.
  • both T14 and T30 increase ⁇ 7- nAChR levels in the membrane compartment.
  • higher amounts of high molecular weight aggregated species of the ⁇ 7-nAChR were observed for T30 incubated cells as compared with controls (Fig. 6B, empty arrowheads).
  • T30 peptide interacts with both the active ligand-binding site and the allosteric modulatory site on the receptor, whereas T14 peptide only interacts with one receptor binding site.
  • T30 peptide induces upregulation of ⁇ 7-nAChR mRNA and protein expression to a greater degree than does T14 peptide.
  • T30 peptide stimulates both AChE and BuChE upregulation and secretion, whereas T14 peptide exposure has no effect on BuChE release and a less potent effect on AChE upregulation.
  • T30 peptide has a greater effect on astroglial cell proliferation at low doses than does T14 peptide.
  • T30 peptide being a more physiologically active and relevant peptide than T14 peptide, just as A ⁇ 42 is more potent and physiologically relevant than the shorter APP peptides (A ⁇ 40, A ⁇ 16, etc) in the pathology of AD.

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Abstract

L'invention concerne le fragment de l'acide aminé C-terminal (30) de l'isoforme T de l'acétylcholinestérase qui présente une activité biologique conforme à un rôle dans l'étiologie d'une maladie neurodégénérative, en particulier la maladie d'Alzheimer, la maladie de Parkinson et une maladie des motoneurones. L'invention concerne également l'utilisation de ce polypeptide (polypeptide T30) à la fois en tant qu'outil de criblage dans des analyses pour la thérapeutique et en tant que marqueur de diagnostic pour une maladie neurodégénérative.
PCT/GB2008/050326 2007-05-04 2008-05-06 Fragment c-terminal biologiquement actif de l'acétylcholinestérase Ceased WO2008135790A1 (fr)

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CN107531796A (zh) * 2015-03-27 2018-01-02 神经生物有限公司 识别AChE的T14肽的抗体
CN108699541A (zh) * 2016-01-28 2018-10-23 神经生物有限公司 癌症
WO2025082212A1 (fr) * 2023-10-17 2025-04-24 合肥科生景肽生物科技有限公司 Banque de peptides cycliques à des fins d'exposition sur phage construite sur la base d'un échafaudage peptidique cyclique naturel

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EP4116415A1 (fr) * 2014-11-26 2023-01-11 Neuro-Bio Ltd Troubles neurodégénératifs
GB2539161A (en) * 2014-11-26 2016-12-14 Neuro-Bio Ltd Neurodegenerative disorders
KR20170104457A (ko) * 2014-11-26 2017-09-15 뉴로-바이오 엘티디 신경변성 장애
WO2016083809A1 (fr) * 2014-11-26 2016-06-02 Neuro-Bio Ltd Troubles neurodégénératifs
JP2018506506A (ja) * 2014-11-26 2018-03-08 ニューロ−バイオ リミテッドＮｅｕｒｏ−Ｂｉｏ Ｌｔｄ 神経変性障害
KR102489620B1 (ko) 2014-11-26 2023-01-16 뉴로-바이오 엘티디 신경변성 장애
RU2707191C2 (ru) * 2014-11-26 2019-11-25 Нейро-Био Лтд Нейродегенеративные расстройства
US10729749B2 (en) 2014-11-26 2020-08-04 Neuro-Bio Ltd. Neurodegenerative disorders
CN114410606A (zh) * 2014-11-26 2022-04-29 神经生物有限公司 神经变性障碍
CN107531796A (zh) * 2015-03-27 2018-01-02 神经生物有限公司 识别AChE的T14肽的抗体
EP3407977B1 (fr) * 2016-01-28 2022-10-19 Neuro-Bio Ltd Méthodes de diagnostic et de prognostic du cancer et des maladies métastatiques, ainsi que biomarqueurs de ces affections
CN108699541A (zh) * 2016-01-28 2018-10-23 神经生物有限公司 癌症
WO2025082212A1 (fr) * 2023-10-17 2025-04-24 合肥科生景肽生物科技有限公司 Banque de peptides cycliques à des fins d'exposition sur phage construite sur la base d'un échafaudage peptidique cyclique naturel

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