WO1994009808A1 - Substances ayant l'effet promoteur de croissance de la proteine precurseur d'amyloïde - Google Patents

Substances ayant l'effet promoteur de croissance de la proteine precurseur d'amyloïde Download PDF

Info

Publication number
WO1994009808A1
WO1994009808A1 PCT/US1992/009070 US9209070W WO9409808A1 WO 1994009808 A1 WO1994009808 A1 WO 1994009808A1 US 9209070 W US9209070 W US 9209070W WO 9409808 A1 WO9409808 A1 WO 9409808A1
Authority
WO
WIPO (PCT)
Prior art keywords
peptide
app
drug
sequence
growth
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/US1992/009070
Other languages
English (en)
Inventor
Tsunao Saitoh
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
University of California Berkeley
University of California San Diego UCSD
Original Assignee
University of California Berkeley
University of California San Diego UCSD
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by University of California Berkeley, University of California San Diego UCSD filed Critical University of California Berkeley
Priority to PCT/US1992/009070 priority Critical patent/WO1994009808A1/fr
Priority to AU28951/92A priority patent/AU2895192A/en
Publication of WO1994009808A1 publication Critical patent/WO1994009808A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • 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
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/54—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one sulfur as the ring hetero atoms, e.g. sulthiame
    • 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
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4711—Alzheimer's disease; Amyloid plaque core protein
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/08—Tripeptides
    • C07K5/0815—Tripeptides with the first amino acid being basic
    • C07K5/0817—Tripeptides with the first amino acid being basic the first amino acid being Arg
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/10—Tetrapeptides
    • C07K5/1019—Tetrapeptides with the first amino acid being basic
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/28—Neurological disorders
    • G01N2800/2814—Dementia; Cognitive disorders
    • G01N2800/2821—Alzheimer

Definitions

  • the present invention relates to the field of proteins associated with neuronal degeneration and growth. More particularly, the present invention relates to short peptide sequences that possess homology with regions within the secreted form of the amyloid ⁇ -protein precursor compound that does not contain a Kunitz-type protease inhibitor domain (sAPP-695) that appears largely responsible for APP's growth promoting effects.
  • sAPP-695 Kunitz-type protease inhibitor domain
  • AD Alzheimer's Disease
  • HSHWA-D hereditary cerebral hemorrhage amyloidosis Dutch type
  • Icelandic type hereditary cerebral hemorrhage amyloidosis Dutch type
  • HCVWA-I sporadic cerebral amyloid angiopathies
  • CAA's sporadic cerebral amyloid angiopathies
  • ARA's age-related amyloidoses
  • amyloid proteins are a major component of the cerebrovascular amyloid deposits, Glenner, et al., Biochem. Biophvs. Res. Coromun.. 122:1131-1135 (1984), and the core of neuritic plaques. Masters, et al., EMBO J. , 4:2757-2763 (1989); Selkoe, et al., J. Neurochem.. 146:1820-1834 (1986), the latter of which is believed to be the hallmark of the pathology found in the brain tissue of patients afflicted with Alzheimer's disease (AD). It is believed that the central pathological theme exhibited by these diseases is an apparently irreversible central nervous system neuronal degeneration or cell death.
  • AD Alzheimer's disease
  • central nervous system neurons undergo degeneration.
  • ischemia caused by physical or chemical assaults to the central nervous system
  • hypoxia where oxygen is cut off from the tissue by either chemical or physical intervention
  • free radical attack on the tissues apparently due to metabolism of various chemicals or the normal aging process, to name but a few.
  • the primary plaque component in each has been identified as a protein subunit of about 4.5 kD having the ability to aggregate.
  • This protein subunit is variously referred to as the amyloid ⁇ -protein, 0-amyloid protein, or as amyloid A4, and is herein referred to as "A4.”
  • A4 is believed to be a degradation product from ⁇ -amyloid precursor protein (as used herein, "APP"). Kang, et al., Nature. 325:733-736 (1987).
  • APP appears as a triplet of polypeptides of molecular weight 110 to 130 kD on Western Blot and in immunoprecipitation. Siman, et al., J. Bio. Chem.. 265: 3836- 3843 (1990) . APP is a membrane-spanning protein, (Kang, et al.. Nature. 325:733-736 (1987); Tanzi, et al.. Science. 235:880-884 (1987); Robakis, et al., Proc. Natl. Acad. Sci. USA.
  • APP-563, APP-751 and APP-770 contain a domain showing a strong homology with protease inhibitors of the Kunitz type (KPI) (Ponte, et al.. Nature. 331:525-527 (1988); Tanzi, et al., Nature. 331:528-530 (1988); Kitaguchi, et al., Nature. 331:530-532 (1988); De Sauvage, et al., Science. 245:651-653 (1989); Golde, et. al., Neuron. 4:253-267 (1990)) while the other two forms (APP-695 and APP-714) do not.
  • KPI Kunitz type
  • sAPP secreted forms of APP
  • sAPP-695 The secreted form of the 695 amino acid protein is referred to herein as "sAPP-695".
  • protease nexin-2 referred to 15 herein as “PN-2”
  • PN-2 protease nexin-2
  • PN-2 protease nexin-2
  • peptides or drugs to promote the healing of neurons that have undergone degeneration.
  • a need remains to prevent pathological changes to neurons that are caused by the above- mentioned diseases, as well as others.
  • BnmiwT- ⁇ e%f the Invention provides an isolated peptide smaller than a native amyloid precursor protein (APP) that retains at least some neuronal growth promoting effect of APP, comprising at least five consecutive amino acid residues with side-chain polarities corresponding to the side-chain polarities of the sequence RERMS (SEQ ID NO:l).
  • the peptide includes the sequence REKVT (SEQ ID NO:18).
  • the peptide includes the sequence RERMS (SEQ ID NO:l) .
  • the peptide has 316 amino acid residues or less, still more preferably it has 150 amino acid residues or less, even more preferably 40 amino acid residues or less, even more preferably seventeen amino acid residues or less, and most preferably five amino acid residues or less.
  • particularly preferred embodiments include a 40-mer corresponding to residues 296-335 of APP, a 17-mer corresponding to residues 319-335 of APP, and a 5-mer with side-chain polarities corresponding to the side-chain polarities of the sequence RERMS (SEQ ID N0:1).
  • an especially preferred 5-mer corresponds to residues 328-332 of APP.
  • Another aspect of the present invention provides a non-peptide drug effective in promoting neuronal growth.
  • This non-peptide drug includes a plurality of functional groups.
  • the functional groups have a polarity, electron distribution and bond length corresponding to the polarity, electron distribution and bond lengths of the side-chains in the peptide RERMS (SEQ ID NO:l).
  • the drug can be produced by a method described below in which structural data is generated on a peptide that has neuronal growth-promoting activity and contains an amino acid sequence having a RERMS (SEQ ID NO:l) sequence or a RERMS-like sequence that has side-chain polarities corresponding to the side-chain polarities of the sequence RERMS, the peptide also containing a plurality of additional amino acids from the sequence of human amyloid precursor protein surrounding the RERMS sequence located therein.
  • the invention also includes a drug comprising the non-peptide drug or the peptide as described above.
  • the drug is capable of crossing the blood-brain barrier of the central nervous system of an animal.
  • the peptide or non-peptide drug has a hydrophobic moiety attached to a terminal position of the drug in a manner such that the hydrophobic moiety will not interfere with the activity of the drug.
  • Yet another aspect of the present invention provides a method of promoting the regeneration of damaged neurons in vivo in a mammal.
  • This method includes identifying a mammal having damaged neurons, and administering a peptide or non-peptide drug as described above to the mammal.
  • the damaged neurons are in the central nervous system (CNS) of the mammal.
  • the invention also provides a method of treating a condition associated with cerebral deposition of the amyloid / 9-protein in a human patient.
  • This method includes identifying a patient having the condition, and administering a peptide or non-peptide drug as described above to the mammal.
  • the condition is Alzheimer's Disease (AD) , and preferably, the patient has not yet developed complete Alzheimer's Disease symptoms.
  • AD Alzheimer's Disease
  • the invention provides a method of treating a neurological condition characterized by damage to neurons.
  • Many such conditions can be treated, including ischemic neurological damage, hypoxic neurological damage, denervation following injury or trauma to the CNS and glutamate toxicity.
  • the method includes identifying a patient having the neurological condition, and administering a peptide or non-peptide drug as described above.
  • a neuroprotectant drug is also administered to the patient.
  • the neuroprotectant drug can be any of many such drugs, including MK 801, other glutamate antagonists, an inhibitor of calpain, chlorpromazine and trifluoperazine.
  • the invention includes the peptide or non-peptide drugs described above for use as an agent to promote neuronal regeneration of neurons of the central nervous system in vivo in an animal, including the use for increasing the memory- retention ability of a mammal.
  • the peptide or non-peptide is also provided for use as an agent to promote neuronal regeneration of central nervous system neurons in patients who have been subject to an ischemic or hypoxic event.
  • Yet another aspect of the present invention is a method of increasing the memory-retention ability of a mammal.
  • a peptide or non-peptide drug as described above is administered to the mammal in a manner such that the peptide or non-peptide enters the central nervous system of the mammal.
  • the peptide or non-peptide can be administered by any of a variety of administration routes, including intramuscular injection, intravenous injection, intrathecal injection, direct infusion into the central nervous system and oral administration.
  • Still one more aspect of the invention provides a growth assay for testing the neuronal growth-promoting activity of a chemical substance.
  • This assay includes the step of adding a measured quantity of a the substance to a culture of a fibroblast cell line that is deficient in its secretion of
  • the cell line should have a substantially slower growth rate than a normal fibroblast cell line not having the deficiency.
  • the assay then includes measuring the growth rate of the culture containing the substance and measuring the growth rate of the culture in the absence of the substance.
  • the growth rate of the culture containing the substance with the growth rate of the culture in the absence of the substance is then compared.
  • An increase in the growth rate of the culture containing the substance relative to the growth rate of the culture in the absence of the substance indicates that the substance has neuronal growth-promoting activity.
  • the fibroblast cell line Although many cell lines can be used, the fibroblast cell line
  • A-l derived from parent cell line AG2804 is one preferred cell line for use in the assay.
  • Cell line B103 is another such preferred cell line.
  • the invention includes a method of designing non-peptide drugs for use as neuronal growth promotion agents.
  • This method includes generating structural data on a peptide that has neuronal growth-promoting activity and contains an amino acid sequence having a RERMS (SEQ ID NO:l) sequence or a RERMS-like sequence that has side-chain polarities corresponding to the side-chain polarities of the sequence RERMS, the peptide also containing a plurality of additional amino acids from the sequence of human amyloid precursor protein surrounding the RERMS sequence located therein.
  • the method also includes determining energetically stable conformations of the peptide by comparing the structural data to structural data of a model protein or peptide using molecular dynamics calculations.
  • the model peptide protein has substantial homology to SEQ ID NO:8. Further steps of the method are determining the active domain in the peptide, calculating the electrostatic potential of the active domain, and designing a low molecular weight non- peptide compound that has a structure that has an energetically stable conformation and matches the electrostatic potential, as determined in the foregoing steps. Finally, the non-peptide compound is synthesized.
  • the step of determining the active domain can be performed by producing variants of the peptide having an amino acid substitution in the RERMS or RERMS-like sequence thereof, such as in the additional amino acids from the sequence of human amyloid precursor protein surrounding the RERMS sequence. Thus, this determining step can also include determining which of the variants provide neuronal growth-promoting activity.
  • the structural data of the peptide is generated by a method such as nuclear magnetic resonance (NMR) , X-ray crystallography, circular dichroism, or small angle neutron scattering (SANS) .
  • the generating step can preferably include the use of NMR.
  • the comparing step can use glucose-6-phosphate isomerase as the model compound.
  • active and inactive substitution parameters can be derived from a growth assay as is described above.
  • the calculating step can include the use of a calculation protocol such as MNDO, AMI or PM3.
  • the designing step can include the steps of obtaining relative configurations of the RERMS or RERMS-like sequence and maintaining the relative configuration in order to design a hydrocarbon or substituted hydrocarbon skeleton that will maintain such relative configuration of amino acids, substituting homologous model moieties onto the skeleton for the original amino acids to produce a model molecule, calculating the structure and distribution of electrons on the model molecule to produce a structural and distributional result, comparing the structural and distributional result with that for a known molecule that has been previously modeled, repeating the substituting, calculating and comparing steps for a plurality of homologous model moieties, determining the optimum homologous moiety for replacement of the homologous model moieties, and synthesizing the molecule that comprises the optimum homologous moieties substituted onto the skeleton.
  • non- peptide drug is also made capable of crossing the blood-brain barrier in the central nervous system of an animal, such as by adding a hydrophobic moiety to a terminal position of the drug and in a manner such that said hydrophobic moiety will not interfere with the activity of said drug.
  • compositions for the treatment of a medical condition associated with neuronal degeneration in the central nervous system of a mammal includes a peptide or non-peptide drug as described above in an amount effective to promote the growth or regeneration of neurons in vivo together with a pharmaceutically acceptable carrier, filler, or excipient.
  • the drug can be present in the composition in an amount effective to treat a condition associated with cerebral deposition of amyloid ⁇ -protein, a hypoxic condition of neurons in the central nervous system, an ischemic condition of neurons in the central nervous system and a condition affecting memory retention.
  • the composition also includes a neuroprotectant drug, such as MK 801, other glutamate antagonists, an inhibitor of calpain, chlorpromazine and trifluoperazine.
  • compositions for increasing the memory-retaining ability of a mammal.
  • the composition includes a peptide or non-peptide drug as described above in an amount effective to increase the memory-retaining abilities of the mammal together with a pharmaceutically acceptable carrier, filler, or excipient.
  • the invention also includes an antagonist of the neuronal growth-promoting activity of amyloid precursor protein, such as a peptide containing the sequence RMSQ (SEQ ID NO:2).
  • Such fragments include a 40-mer, consisting of 40 amino acids, residues 296-335 of APP, and a 17-mer, consisting of 17 amino acids, residues 319-335 of APP and an 11-mer, consisting of 11 amino acids, residues 325-335 of APP. See, e.g., Kang et al. supra for the full sequence of APP.
  • Each of the fragments within the series possesses the 5-mer sequence of R-E-R-M-S (SEQ ID NO:l) .
  • the 5-mer possesses a substantial amount of the neuronal growth promoting activity as native sAPP-695, however, less activity than either the 40-mer, the 17-mer or the 11-mer.
  • RERMS sequence of five amino acids
  • APP328-332 the sequence of five amino acids, RERMS (SEQ ID NO:l) (APP328-332)
  • RERMS sequence of five amino acids
  • peptides of APP in which the RERMS (SEQ ID NO:l) sequence i ⁇ deleted or reversed do not possess growth promoting activity.
  • RMSQ amino acid peptide RMSQ (SEQ ID NO:2), which corresponds to residues 330-333 of APP, and which partially overlaps the C-terminal side of the active sequence RERMS (SEQ ID NO:l)
  • Peptides which contain the active sequence RERMS (SEQ ID NO:l) of the present invention are useful to promote the growth of neurons in vitro and in vivo .
  • Such fibroblast's growth may be promoted by addition of exogenous APP.
  • the peptides that we have discovered that possess the RERMS (SEQ ID NO:l) promote growth of the A-l cell line.
  • fibroblasts for a simple bioassay of the growth promoting effect of our APP fragments.
  • Such cell lines are prepared and maintained as described previously. See Saitoh, et al., Cell. 58:615-622 (1989).
  • DMEM Dulbecco's modified Eagle's medium
  • FCS fetal calf serum
  • A-l cells were maintained in the same way with the exception that the media contained the conditioned media (CM) from AG2804 cell culture (20% v/v) .
  • CM CM from AG2804 cells
  • Al cells used for the growth assay were kept in the regular media (DMEM/10% FCS) without CM for one week before the assay.
  • APP appears to possess sites that act as agonists and antagonists in neuronal growth promotion. This finding is useful in further delineating the biological activity of sAPP-695 in physiological and pathological conditions.
  • Roch, et al., J. Biol. Chem.. 267:2214-2221 (1992) we showed that both KB75 (APP residues 20-591) and 40-mer (APP296-335) were active in our A- 1 cell growth assay.
  • KB75 APP residues 20-591
  • 40-mer APP296-335
  • Peptides corresponding to all or part of APP296-335 were synthesized using a Rainin PS3 peptide synthesizer and purified on a c-18 reverse-phase HPLC column.
  • the amino acid sequences of the synthetic peptides synthesized and used are detailed in Table I. Each peptide was recovered in 40% acetonitrile. The acetonitrile was eliminated by evaporation under vacuum, and the peptide was resolubilized in 50 mM NaHC0 3 (pH 7.4).
  • KB75 that encodes for APP residues 20 through 591
  • a plasmid KB755 which encodes APP residues 20 through 591 without residues 306-335.
  • the construct KB75 ⁇ was designed to enable us to see more directly whether the domain within residues 306 through 335 is the only active site of sAPP-695 or if there is an additional active site. As will be appreciated, this is enabled since residues 306 through 335 contains the majority of the 40-mer sequence and all of the 17-mer and 5-mer. Thus, since the 40-mer, 17- mer, and 5-mer are all known to be active, their elimination from KB755 allows us to determine if there are other regions with growth promoting activity.
  • KB75 and KB755 were obtained through the bacterial expression and protein purification procedures, which are described in greater detail in Roch, et al., J. Biol. Chem.. 267:2214-2221 (1992), in connection with KB75, which encodes for the amino acids Val-20 to Ile-591 of sAPP-695.
  • the strategy for the construction of the plasmid pKB75 ⁇ is shown in Figure 7.
  • the oligonucleotides used as primers in the PCR reaction were synthesized in a PCR-mate DNA synthesizer (Applied Biosystems) and purified by the OPC system.
  • the sequence of the 5' primer was 5'- GACAGTGTCACTCGAGAGAGAATGGGAAGAGGCAGAA-3' (SEQ ID NO:3) and the 3' primer was 5•-GGACTGAGTCCTCGAGCTAGATCTCCTCCGTCTTGAT AIT ⁇ S' (SEQ ID NO:4).
  • the template for the PCR reaction was the 2.4 kb Smal-Xmnl fragment of APP-695 cDNA.
  • the reaction was carried out in 100 ⁇ l containing 500 ng of template DNA, 1 ⁇ M of each primer, 200 ⁇ M of each dNTP and 1.5 units of enzyme AmpliTaq.
  • the sample was brought to a temperature of 94 "C for 3 minutes, followed by 38 cycles, following the protocol: 94 ⁇ C for 30 seconds, 55°C for 1 minute, 72 ⁇ C for 1 minute. After the last cycle, a final extension step was performed at 72 ⁇ C for 7 minutes.
  • the PCR product (800 bp in length) was finally purified by electrophoresis in 1% agarose gel, binding to a DEAE- cellulose membrane, elution in 1M NaCl at 65 ⁇ C, and ethanol precipitation.
  • the purified PCR product was then digested with Xhol restriction enzyme, and ligated into the plasmid pKB755 previously digested with Xhol and treated with canine intestine alkaline phosphatase.
  • Competent E. coli MC1061 cells were transformed with the ligation mixtures and clones were selected for ampicillin resistance.
  • the size and orientation of the insert were determined by restriction analysis, using different enzymes having asymmetrical restriction sites.
  • anti-GId antibody which is a rabbit polyclonal antibody against the peptide 175-186 of APP (Saitoh, et al..
  • Cells at 70-80% confluence were detached from the substratum by incubating in phosphate buffered saline (PBS) containing ImM EDTA.
  • PBS phosphate buffered saline
  • the detached cells were harvested, centrifuged down and resuspended in DMEM/10% FCS at the density of 5,000 cells/ml.
  • the cell suspension was then seeded in six-well plates (2 ml/well) and the plates were kept at 37 ⁇ C in the C0 2 incubator for one hour.
  • the relevant peptide solution in DMEM 100 ⁇ l was then added, the same volume of DMEM without peptide was added in control wells.
  • the plates were kept at 37 ⁇ C in the C0 2 incubator for three days and the cell number in each well was determined using a Coulter cell counter, model ZF (Coulter Electronics, Inc., Hialeah, FL) . The results were expressed as the percent increase in the cell number compared to the number of the cells seeded. All the growth assays were done in triplicate.
  • reverse-sequence 17-mer M-V-Q-S-M-R-E- R-H-K-A-E-L-R-E-K-A (SEQ ID NO:17).
  • the reverse sequence 17- mer was prepared in accordance with Example II and tested in accordance with Example V. As will be seen in Figure 3, no activity was detectable in the reverse sequence 17-mer.
  • incorporatioh of tritiated thymidine into the cells was determined.
  • the growth assay of Example V was followed, with the additional step of measuring the tritiated thymidine's incorporation. This was accomplished by washing the cells after three days incubation with DMEM and then incubating the cells in DMEM containing [ 3 H]thymidine (Amersham Co., 5 ⁇ Ci/ml, 1 ml/well) for three hours at 37°C in the C0 2 incubator.
  • the cells were then fixed with methanol/acetic acid (1:3 v/v) and washed with ice-cold 10% TC (0.5 ml x 5 times). The radioactivity remained in the cells was extracted by overnight incubation in 0.5 ml of 1 N NaOH and was counted in 10 ml of scintillation cocktail.
  • recombinant protein KB15S was obtained through the bacterial expression and protein purification procedures, which were exactly the same as employed for the preparation of KB75.
  • Immunoblotting with anti-GID antibody detected the expression of KB755 in the partially purified bacterial lysate, with a predicted difference in the molecular weight from that of KB75.
  • the peptide was further purified through heparin-agarose column and size-exclusion HPLC, to give an apparently single band on Coomassie blue staining of the SDS-PAGE gel (data not shown) .
  • KB755 did not possess any growth promoting activity or effect with the A-l cell line. This result indicates that the deleted sequence Thr-306 to Met-335, in KB755, is essential for the activity of the APP and its fragments.
  • the wells were coated with the peptide according to the procedures described by Schubert, et al.. Neuron. 3:689-694 (1989) and the adhesion of cells to the precoated wells was determined as described above. All the assays were done in triplicate. The results were expressed as the percentage of cells recovered in the media compared to the total number of cells recovered.
  • KB755 The lack of the activity of KB755 in the present study (Fig. 12) is a strong evidence indicating that the deleted sequence in KB755 (APP306-335, Kang sequence) included the only one active site of sAPP-695 in the A-l cell bioassay.
  • the growth-promoting activity of this site was represented by the 5 amino acids sequence RERMS (APP residues 328-332) SEQ ID NO:l (Fig. 1).
  • the concentration of the peptide needed for the significant effect was approximately tenfold higher for RERMS than for either the 11-mer or 17-mer, suggesting that the amino acid sequence in the vicinity was necessary for the full activity.
  • the reverse-sequence 17-mer had no activity (Fig.
  • the RERMS (SEQ ID NO:l) motif and its N-terminal side amino acid sequence have some interesting characteristics.
  • the sequence RERMS (SEQ ID NO:l) is unique for APP; we found no match in the peptide sequences so far registered in GeneBank.
  • it is within one of the evolutionary highly conserved regions of APP (Rosen, et al. 1989). Exactly the same sequence as 40-mer is found in mammalian APPs (rat, mouse, and monkey).
  • APP-like there is a sequence REKVT (SEQ ID NO:18) (residues 423-427; Rosen, et al.
  • APP binds to heparin-sulfate proteoglycan (Narindrasorasak, et al., J. Biol. Chem.. 266:12878-12883 (1991)), which is supposed to be involved in cell- extracellular matrix interaction.
  • APP is one of the heparin-binding proteins (Schubert, et al., Proc. Natl. Acad. Sci. USA f 86:2066-2069 (1989)), the reason why we used heparin-agarose column to purify it from the bacterial lysate (Roch, et al., J. Biol. Chem.. 267:2214- 2221 (1992)) .
  • sequence APP306- 335 may contribute to the heparin-binding capacity of sAPP-695 in its native form of the molecule, the growth-promoting activity of the domain in itself had little to do with the heparin-binding capacity.
  • sequence APP305-335 contained the only active site of sAPP-695 and that the activity was represented by the 5 amino acid sequence RERMS (SEQ ID NO:l) (APP328-332) .
  • the biochemical basis for the activity is still unknown and we suppose the presence of a cell-surface molecule(s) recognized by this domain of sAPP.
  • mice monoclonal anti-APP (Clone 22C11, Boehringer
  • Sections were the incubated in a mixture of FITC-conjugated horse anti-mouse IgG (1:100), followed by Avidin D Texas red (1:100; Vector Laboratories, Inc., Burlingame, CA) . Imaging and Analysis The double-labeled sections were transferred to SuperFrost plus slides (Fisher Labs, Tustin, CA) and mounted under glass cover slips with anti-fading media containing 4% n-propyl gallate (Sigma Chemical Company, St. Louis, MO) . The sections were studied with the Bio-Rad MRC-600 laser scanning microscope mounted on an Axiovert Zeiss microscope. This system permits the simultaneous analysis of double- immunolabeled samples in the same optical plane.
  • anti-APP-immunolabeled terminals were slightly larger than the average terminal immunolabeled with anti- GAP43.
  • anti-APP displayed a stronger immunostaining intensity in the pyramidal neurons of the frontal cortex as compared to controls ( Figure 13), while anti-GAP43 immunoreactivity was decreased in the cell body of the neurons, but increased in the neuritic processes.
  • a prominent finding in the AD cases was the presence of intensely anti- GAP43 and anti-APP immunoreactive abnormal neurites in the plaques ( Figure 14) .
  • APP upregulation in AD appears to be associated with the aberrant sprouting response in the brain as a consequence of the synapse loss and neuro-degeneration.
  • 0/A4 amyloid deposition is the result of.the upregulation and abnormal processing of APP, but not the cause of the neuropil damage in the first place.
  • the forebrains of eight 1-day-old (PI) Sprague-Dawley rats were obtained and fixed by immersion in 2% paraformaldehyde in phosphate buffered saline (pH 7.4) for 72 hrs at 4 ⁇ C.
  • the tissue was serially sectioned at 40 ⁇ m with the Dosaka microslicer.
  • Antibodies The mouse monoclonal anti-APP (Clone 22C11, Boehringer- Mannheim, Indianapolis, IN) was used to label the neonatal rat brain sections.
  • the antibody was obtained by immunizing mice with purified recombinant APP fusion protein.
  • the antibody was purified from ascites fluid by ion-exchange chromatography and tested elsewhere.
  • the polyclonal antibody against GAP-43 was produced by immunization of rabbits with a gel purified protein as described previously. The antibody specificity and characteristics have been described. Further double- immunohistochemical analyses were done with the rabbit polyclonal antibody that recognizes phosphorylated epitopes on medium and heavy subunits of neurofilament proteins in axons (Sigma Chemical Company, St. Louis, MO) , with a rabbit polyclonal antibody against glial fibrillary acidic protein (GFAP, Biogenex, San Ramon, CA) and with the mouse monoclonal antibody against synaptophysin (SY 38, Boehringer-Mannheim) . Double Immonolabeling and Laser Confocal Imaging of Neurites in the Neonatal Rat Brain
  • Sections were the incubated in a mixture of FITC- conjugated horse anti-louse IgG (1:70) and biotinylated goat anti-rabbit IgG (1:100), followed by Avidin D Texas red
  • APP was co-localized with GAP-43 positive neuritic fibers (Figs. 16C, 17B, C) .
  • APP was also occasionally co-localized with GAP-43 positive horizontal fibers (Fig. 17A) .
  • APP immunoreactive neuritic processes were ramified in their distal portion (Fig. 17B) and contained anti- neurofilament immunoreactivity (Fig. 17D) , although they were anti-GAP negative.
  • the present study showed colocalization of APP immunoreactivity in GAP43-immunolabeled neurites in the neonatal rat neocortex and hippocampus. These neuritic processes corresponded to long fibers originating in neurons distributed in the inner layers. Previous studies in the neonatal rat brain have shown that these processes correspond to growth cones that eventually will establish the cortical microcircuits. Moreover, biochemical studies have shown that neonatal rat brain contains high levels of APP, identified in the Western blot with an anti-C terminal antibody as a band at 110 to 130 kD.
  • APP mediates neuronal cell-cell and cell surface adhesion similar to N-CAM in sympathetic cells.
  • APP is produced in a secretory and non-secretory form, in different cell lines.
  • the human glioma Bu-17 cells produces only the non-secretory form of APP, and in these cells APP immunoreactivity is accumulated in the growth cones, suggesting that the non-released form of APP may play a role in process growth through regulating cell adhesion.
  • the N-terminal region of the APP molecule, with KPI domain has been identified as PN-II, a potent inhibitor of chymotrypsin, coagulation cascade proteases, and forms stable complexes with EGF binding proteins and the gamma subunit of
  • AD APP is accumulated in abnormal neuritic processes in the plaques that are enriched with GAP-43.
  • APP upregulation or abnormal processing in AD might be associated with an abnormal growth response in the brain as a consequence of the ongoing synaptic pathology.
  • AD Alzheimer's disease
  • GAP43 growth associated protein
  • the ⁇ -protein is derived from a larger precursor protein, APP.
  • Antibodies against specific regions of the APP molecule immunolabel the plaque dystrophic neurites and the APP immunoreactivity of the hippocampal neurons is increased in AD.
  • APP mediates neuronal cell-cell and cell surface adhesion similar to neural cell adhesion molecule (N- CAM) .
  • APP also promotes fibroblast growth and a fragment corresponding to the A4 region enhances the survival and neurite extension of hippocampal neurons in vitro .
  • the N terminal region of the APP molecule with the Kunitz protease inhibitor (KPI) domain, has been identified as PN-II, a protease inhibitor probably involved in neuritic outgrowth.
  • KPI Kunitz protease inhibitor
  • PN-2 participates in regulation of neurite extension (Van Nostrand, et al.. Science, 248:745-748 (1989); Oltersdorf, etal.. Nature. 341:144-147 (1989)), blood coagulation processes (Cole, et al., Biochem. Biophvs. Res. Commun.. 170, 288-295 (1990); Van Nostrand, et al., 1990; Smith, et al., Science. 248:745-748 (1990)) and in wound- healing process (Cunningham, et al., Brain Res. Rev..
  • sAPP-695 is secreted from cells and has a growth promoting effect on fibroblasts. Saitoh, et al., Cell. 58:615-622 (1989). Consequently, in connection with our discovery that only a small portion of the amino acid sequence of sAPP-695 is responsible for the growth enhancement in fibroblasts, we have also discovered that fragments of sAPP-695 containing the RERMS sequence, discussed above, exhibit a profound ability to enhance the growth of neurons.
  • rat CNS neuroblastoma cell line that responds to APP by extending the neurites.
  • This allows a relatively simple assay to identify peptides that participate in neuron growth promotion. While our primary assay employs this neuroblastoma cell line, we have also used primary neuronal culture successfully to demonstrate APP activity. Further, we have tested all of the peptides discussed above incorporating the RERMS sequence in connection with their ability to participate in neuron regeneration.
  • sAPP secreted forms of APP
  • B-103 clonal CNS neuronal line B-103 that does not synthesize or secrete APP from Dr. Schubert, at the Salk Institute for Biological Studies.
  • B-103 cells were plated at low density in a synthetic serum free medium, neurite outgrowth was promoted by the conditioned medium from APP-695 or APP-751 over-producing cells and also by the bacteria produced sAPP-695 (named KB75) and sAPP-751 (named Nex II) .
  • the 17-mer was iodinated with [ 15 I]Bolton-Hunter reagent (Amersham Co., 2000 Cl/mmol, 500 ⁇ Cl) according to the manufacture's instructions.
  • the peptide solution (10 ⁇ g in 10- ⁇ l of 0.1 M NaHC0 3 buffer, pH 8.5) was incubated with the dried reagent at 4*C overnight.
  • the reaction was quenched with glycine (100 ⁇ l of 1 mg/ml solution of H 2 0) and the iodinated peptide was separated from the unincorporated reagent through Sephadx G15 column (1 cm 2 x 25 cm) saturated with PBS containing 0.15% gelatin.
  • the specific activity of the obtained 125 l-17-mer was 40 to 60 Ci/mmol.
  • Binding Assay The cells in the late log phase were harvested in PBS, and spun down at 1,200 g for 5 min. Thereafter, the cells were suspended in DMEM/5%FCS. The suspension (400,000 cells/ml) was seeded in 24-well plate (0.5 ml/well) and the plate was kept at 37°C in a C0 2 incubator for three hours. The plate was then transferred on top of ice and the media was immediately replaced with ice-cold PBS containing 0.15% gelatin (PBSG) . After 30 min, the binding reaction was started by replacing the PBSG with fresh, ice-cold PBSG containing 125 I-l7-mer and various peptides as indicated above. The reaction volume was 0.25 ml.
  • the binding media was aspirated and the cell layer was washed three times with 0.5 ml of ice-cold PBSG.
  • the radioactivity remaining on the cell layer was then recovered in 0.5 ml of the extraction buffer (0.5% Triton X-100, 0.1 M NaHC0 3 pH 7.4) and was counted by 7 - counter. All the binding assays were conducted in triplicate.
  • Table 1 provide ⁇ a summary of the inhibition of the radiolabeled 17-mer in its binding to and with the B-103 cell layer by the indicated peptides.
  • concentration of 125 l-l7-mer was 10 nM.
  • Nonspecific binding was defined as the binding in the presence of 1 ⁇ M unlabeled 17-mer.
  • the figures presented were calculated from the data shown in Figures 3-5 (means 1 SEM of at least three determination each done in triplicate) .
  • PC12 mutant cell line 0141 is susceptible to a high concentration of APP whereas the parent PC12 is not, under the identical conditions.
  • D. Neurotrophic Properties of the APP Moieties Containing the RERMS Sequence in in vivo Rabbit Models Considerable evidence supports the hypothesis that APP is neurotrophic, at least under some circumstances. We have recently demonstrated, as was discussed in greater detail above, that the neurotrophic properties of APP in cell culture are fully preserved in a the 17-mer of the present invention.
  • This 17-mer has been tested on a neuronal cell line (B-103) that does not express APP.
  • Neuroblastoma cultures of B-103 treated with the 17-mer had more neurite-bearing cells, and longer neurite ⁇ , than untreated cells.
  • Other neurotropic factor ⁇ ⁇ uch as NGF and FGF also have been shown to alleviate neuronal loss resulting from some types of injury. If APP is neurotrophic in vivo, then it would be anticipated that administration of the growth-promoting segment of APP might attenuate the neuronal loss of behavioral deficits associated with neuronal injury, such as that accompanying CNS ischemia.
  • Neurologic function was evaluated by two observers blind to animal treatment. Evaluations were conducted at 24 hours and again on the third day following ischemia (experimental day 4) . Animals were classified by the presence or absence of paraplegia. Paraplegic animals, for example, showed more respon ⁇ e to obnoxious ⁇ timuli in the hind limbs and were also totally incontinent. Rabbits that were not paraplegic were either normal or had ⁇ ome motor function in the hind limbs, even if only barely detectable. Bowel and bladder function were variable. If it was difficult to ascertain whether an animal was totally paraplegic, it was classified as partially paraplegic.
  • animal ⁇ were ⁇ acrificed with Euthanol (Schering-Plough, Kenilworth, NJ) . After sacrafice, the spinal cords of the animals were rapidly removed and replaced in fixative (10% phosphate- buffered formalin) for immunohistochemical analysi ⁇ and hi ⁇ tological evaluation.
  • Neurotoxin-induced subcortical le ⁇ ion ⁇ were a ⁇ ociated with increa ⁇ ed APP synthesi ⁇ and turnover in the brain region ⁇ innervated.
  • Thi ⁇ re ⁇ pon ⁇ e wa ⁇ rapid, persisted for several days, and was not a respon ⁇ e to general stres ⁇ but wa ⁇ ⁇ pecific to differentation.
  • APP production rapidly increa ⁇ e ⁇ in brain injury, the rate of turnover al ⁇ o increa ⁇ e ⁇ . Whether thi ⁇ is part of an injury mechanism, a repair mechanism, or unrelated is not apparent.
  • APP promoter contain ⁇ the heat- ⁇ en ⁇ itive element, we believe that the induction of APP ⁇ ynthe ⁇ i ⁇ i ⁇ part of the protective mechanism of cells.
  • a ⁇ will be discussed below, we have recently demonstrated that APP peptides containing the RERMS sequence (SEQ ID N0:1) can support the survival of rat primary cortical neurosis and also can promote the neurite extension of primary culture neurons and neurobla ⁇ toma. The latter might manife ⁇ t an activity to stimulate the sprouting of neurons and to increase the ⁇ ynaptic contact.
  • the reparative activity of 17-mer is entirely consistent with our immunohi ⁇ tochemical analysis of the ischemic tis ⁇ ue to which the 17-mer wa ⁇ admini ⁇ tered. While the 17-mer i ⁇ ineffective in reducing necrotic ti ⁇ ue or number of neuronal lo ⁇ , the 17-mer enhanced the number of anti-tau po ⁇ itive neuronal population. Thi ⁇ re ⁇ ult clearly indicates that the 17-mer has an effect on the plastic reaction of remaining neurons. Indeed, the increased synaptic densities as ⁇ ociated with neuron ⁇ that were treated with the 17-mer i ⁇ further confirming of this mechanism. Thus, the effect of the 17-mer does not appear to be the direct protection of neurons under ischemic stre ⁇ . Rather, the 17-mer appear ⁇ to lead to promote the cell ⁇ remaining after the i ⁇ chemic in ⁇ ult to ⁇ prout and extend neurite ⁇ and expand in ⁇ ynaptic den ⁇ ity, in order to compensate for the lost neuron ⁇ .
  • a ⁇ will be under ⁇ tood, one may combine the two moietie ⁇ in a single administration, or one may reverse the order. However, in either event, the two activities of each moiety act to supplement the activity of the other. Essentially, it appears as though the neuroprotective drug acts to shield the neural cells from the action of ischemic agent or event, yielding a larger number of neurons that survive the ischemia. Further, the peptide, containing, or drug based upon, the RERMS sequence (SEQ ID NO:l) will aid in the promotion and thriving of those neurons that survived.
  • MK-801 i ⁇ an n-methyl-deaspartic acid glutamate antagonist.
  • glutamate antagonists are suitable for u ⁇ e in the pre ⁇ ent invention.
  • a ⁇ well, protein kina ⁇ e antagoni ⁇ t ⁇ and calcium channel blockers have shown similar effect.
  • agents such as trifluoperazine (Stelazine*, Smith, Kline & French) and chlorpromazine
  • APP moieties and analogues based on the 5-mer RERMS sequence SEQ ID NO:l
  • 17-mer of the present invention can induce both morphological and behavioral changes when injected in rat brains.
  • the general protocol that we employed in arriving at thi ⁇ dicosvery was the direct infusion of the 17-mer, a peptide having the rever ⁇ e sequence of 17-mer, or artificial cerebrospinal fluid (CSF) , a ⁇ a control, were infused into 20 month-old rat brain ventricles using a mini osmotic pump.
  • the animals were te ⁇ ted for memory performance u ⁇ ing the Morri ⁇ Water Maze paradigm (MWM) . After the la ⁇ t behavioral te ⁇ t, the animal ⁇ were ⁇ acrificed and their brain morphologically examined.
  • the maze consisted of tank 152 cm in diameter, painted black on the inside and filled with tap water (room temperature) to a depth of 34 cm.
  • the pool was located in the center of a room with numerous extra maze cues (e.g. shelves, tables, the computer) .
  • the ceiling lights were turned off and indirect illumination was provided by 2 lights located on either side of the pool and directed one toward the ceiling, and the other toward the floor. These lights provided diffuse illumination throughout the room so that the extra maze cues were visible from the pool, but did not illuminate the inside of the pool, which wa ⁇ dark, ob ⁇ curing the location of the black platform.
  • the pool wa ⁇ divided into 4 equal quadrants formed by imaginary lines inter ⁇ ecting at the center of the pool, at right angle ⁇ .
  • Such lines in turn intersect the edge of the pool at the arbitrary cardinal start locations called North, South, East, and We ⁇ t.
  • the platform wa ⁇ placed at the center of the South ⁇ west (SW quadrant or the North-East (NE) quadrant.
  • the swim path of the rats, path distance, path length in each quadrant, and escape latency (time to reach the platform) were monitored using a video camera, and videotrack images were digitized and analyzed using a computer program designed for water maze data analysi ⁇ (San Diego Instruments, San Diego, CA) .
  • Each rat was given a block of 4 trials every test day.
  • the start locations were ordered in a semirandom manner.
  • the fir ⁇ t and la ⁇ t trial of each day were started from one of the two location ⁇ farthe ⁇ t from the platform, but the ⁇ tart location of the first trial was never the same on two consecutive day ⁇ .
  • the ⁇ tart location of the second and third trial ⁇ was a random choice between one of the two remaining possibilities.
  • the rats had cannulae implanted into the right lateral ventricle.
  • the cannulae were connected to Alza model 2002 osmotic minipump (Alza Corporation, Palo Alto, CA) placed subcutaneou ⁇ ly in the dor ⁇ al region of the neck/back.
  • Animal ⁇ were infu ⁇ ed either with artificial CSF (saline solution) , with the 17-mer peptide at ImM, or with the peptide of reverse-sequence 71-rem at ImM. Both peptides were di ⁇ olved in artificial CSF a ⁇ vehicle.
  • the infu ⁇ ion wa ⁇ carried out throughout the experiment, at a rate of 0.25 ⁇ l/hr.
  • the rat ⁇ were fir ⁇ t trained for 5 day ⁇ to escape from water onto a platform in the water maze with the visible platform. To avoid the pos ⁇ ibility that the rats use memory cue to reach the platform, its location wa ⁇ changed randomly every day. At the end of the vi ⁇ ible training, the time to reach the platform was not different between the old and young group ⁇ (data'not shown) suggesting that there is no physical, visual, or motivational impairment in the old rats.
  • the rats were then trained for 5 days with the invisible platform which was located at the center of the SW quadrant. The time that the rats required to find the platform did not change significantly during the last 3 days of testing. As an index of the learning performance, we used the mean of the times for animals to locate the invisible platform of the last 3 days. Thi ⁇ mean wa ⁇ significantly higher for the old rats than for the young rates (17.7 s - 8.8 +/ vs 8.5 +/- s p ⁇ 0.003) .
  • the group of old rats showed reduced learning and memory ability when compared to the group of young rat ⁇ . It should be noted that these differences in time could not be attributed to reduced swimming capacities in the old group, becau ⁇ e they performed as well as the young rats in the test with the visible platform.
  • the group assignment was in the order of A-B-C in the triplet containing the best 3 rats.
  • the a ⁇ ignment order was C-B-A.
  • the mean scores of behavior for three groups are similar.
  • the rat ⁇ were re-trained in the water maze fir ⁇ t with a vi ⁇ ible platform and then with an invi ⁇ ible platform located in the center of the SW quadrant (old location) .
  • the invi ⁇ ible platform wa ⁇ moved to the center of the NE quadrant (new location) .
  • the animal ⁇ were ⁇ cored for their path length in the SW quadrant, where the platform used to be present.
  • the ⁇ core of the group A was 249 +/- 133; the score of the group B was 214 +/- 135; and the score of the group C was 258 +/" 185.
  • the differences between these score ⁇ of the 3 group ⁇ did not reach ⁇ tatistical significance (p values > 0.1360).
  • our analysis had only compared the average path length of each group (mean score of the last 3 days of each test) .
  • Thu ⁇ becau ⁇ e each group contained rat ⁇ that ranged from "normal” to "very impaired," an effect of the peptide on a ⁇ ubpopulation of these rats (e.g. only on the normal rat ⁇ , or only on the impaired rat ⁇ ) would not have been detected by analyzing the mean ⁇ of the groups. Therefore, we decided to carry out correlation analysi ⁇ .
  • APP is a neurotrophic factor.
  • APP is required for the function and ⁇ urvival of neurons.
  • trophic factors are found to be effective in diminishing the damage of neurons caused by various insults, and because APP seems to be directly involved in the case of Alzheimer' ⁇ disease, APP or APP-like moietie ⁇ that contain the RERMS sequence should be prime candidates for the treatment of AD and other diseases or events in which neurons are impaired.
  • non-peptidic APP-mimicking drugs to be administered sy ⁇ temically.
  • NMR small angle neutron scattering
  • the NMR ⁇ pectro ⁇ copy is conducted on a fragment of the APP moiety that contains the RERMS ⁇ equence.
  • each of the 5-mer, 11-mer, 17-mer, and 40-mer may advantageou ⁇ ly be studied.
  • Obtaining spectral data for one or more than one fragment allows graphic depictions of natural solution conformational changes as the surrounding amino acids are stacked around the RERMS sequence.
  • 17-mer alone is an ideal candidate for the molecular studie ⁇ , since, it contains all of the activity of the 40-mer and enhanced activity over APP, yet i ⁇ small enough in size to permit reliable and efficient analysi ⁇ .
  • Glucose 6-Phosphate Isomerase may advantageously be used as a model, because it has an overall homology with the 17-mer and X-ray crystallographic analyses have been conducted upon it so that accurate conformational data is available.
  • a composite of the structure of the 17-mer as derived from its NMR spectrum as compared with NMR and X- ray data from Glucose 6-phosphate isomerase may be used. From these structures, an energetically stable conformation can be determined by one of skill in the art using routine molecular dynamic calculation ⁇ . Thereafter, the active domain of the APP moiety containing the RERMS sequence may be further identified through point mutation of the amino acid residues surrounding the active RERMS sequence.
  • such point mutations are conducted upon the 9th through the 14th amino acid residues.
  • Such point mutation ⁇ allow u ⁇ to determine the ⁇ pecific residues where ⁇ ub ⁇ titution create ⁇ a variable activity from the native form.
  • Each of the mutant forms are then analyzed with NMR to further as ⁇ ess the conformational changes produced thereby.
  • electro ⁇ tatic potential ⁇ within the active domain. Electronic di ⁇ tribution within the active domain may be calculated by MNDO, AMI, or PM3 methods. Further, electronic distribution ⁇ are al ⁇ o calculated for the point mutated peptides. From this combined data, the electrostatic potential required for activity within the active domain may be easily derived.
  • the active moieties are substituted with synthetic molecules that posses ⁇ homologous electrical and stearic properties to the original active moieties. Then, through calculating the structure and distribution of electron ⁇ of the compound obtained according to the above procedure ⁇ one may identify the be ⁇ t fit. Compounds corresponding to the fit may be synthesized through routine synthetic chemistry. NMR Spectroscopy
  • NMR magnetic resonance
  • NMR nuclear magnetic resonance
  • the output i ⁇ expressed in terms of hertz (cycle ⁇ per second) .
  • the output provides basically four separate measurements: intensity (which in proton magnetic resonance (PMR) correspond ⁇ to the number of equivalent proton ⁇ ) , chemical shift (the difference in frequency between the excitation of the moiety as referenced to a ⁇ tandard) , width (provide ⁇ an indication of the mobility of peptide) , and coupling constants (measures interactions between nearby magnetic nuclei) .
  • PMR proton magnetic resonance
  • width provide ⁇ an indication of the mobility of peptide
  • coupling constants measures interactions between nearby magnetic nuclei
  • NMR spectra of the point mutated 17-mer provide additional information about the molecular conformation and dynamics cau ⁇ ed by ⁇ uch point mutation ⁇ . Consequently, we are able to develop highly refined molecular conformation information from our NMR spectro ⁇ copy.
  • a detailed discus ⁇ ion of the con ⁇ ideration and calculation ⁇ that are involved in NMR determination of protein and peptide structure is provided in W ® thrich, Science. 243:45-50 (1989).
  • RMD restrained molecular dynamics
  • X-ray cry ⁇ tallography provide ⁇ a comprehen ⁇ ive three dimen ⁇ ional ⁇ tructure that can confirm and integrate these other techniques.
  • NMR i ⁇ generally carried out in ⁇ olution, which eliminates ' the perpetual problem of ⁇ olving often complex crystallization problems.
  • thi ⁇ i ⁇ true in the case of relatively small peptides, where crystallization is virtually impossible. This is especially true in the case of our analyses of the conformational structure of the 5-mer and the 17-mer.
  • RERMS ⁇ equence appear ⁇ indispensable to the growth promoting activity of APP, the RERMS sequence itself contains only 10% of overall activity. We believe that these interactions as ⁇ i ⁇ t in binding and thereby contribute to binding ⁇ pecificity. Thu ⁇ , we have di ⁇ covered that it i ⁇ these interaction ⁇ in conjunction with information obtained from the active ⁇ ite that make the de ⁇ ign of ⁇ pecific promoter molecule ⁇ a po ⁇ ibility. Moreover, this information additionally permits the de ⁇ ign of ⁇ pecific promoter molecule ⁇ for related but nonidentical molecule ⁇ .
  • each member may have only one specific target.
  • succe ⁇ ful rational drug de ⁇ ign ba ⁇ ed on group ⁇ imilaritie ⁇ would provide molecule ⁇ that al ⁇ o interact broadly.
  • a broadly acting drug would interact with any number of receptor ⁇ on the cell from the group the receptor ⁇ that may be responsible for particular actions.
  • a variety of techniques can be used. These technique ⁇ include, circular dichroism, small angle neutron scattering, diffraction methods, including any combination of multiple and single isomorphou ⁇ replacement, single or multiwavelength anomalous scattering methods, molecular replacement methods maximum entropy phasing, solvent-flattening methods and so-called "direct” methods used primarily to solve small-molecule structures.
  • NMR allows one to accurately predict three- dimen ⁇ ional -attribute ⁇ in the interaction.
  • X-ray crystallography may be used in order to generate specific coordinates for each of the non-hydrogen atoms in the complex. Coordinates for the hydrogen atoms can additionally be obtained using neutron ⁇ .
  • X-ray crystallography can be advantageously utilized to determine the molecular conformations exhibited by APP to promote neuron growth. Indeed, we anticipate the use of X-ray crystallography to confirm our conformational studies. For example, we expect that we will turn to larger molecular studies to further identify folding patterns that may either a ⁇ sist or hinder APP in it ⁇ ability to engage the receptor or receptor ⁇ . Moreover, X-ray cry ⁇ tallography will be used in efforts to co-cry ⁇ talize the receptor or receptor ⁇ in conjunction with any of the active peptide ⁇ of APP, a ⁇ they are determined.
  • X-ray cry ⁇ tallography permit ⁇ three dimen ⁇ ional molecular analy ⁇ i ⁇ of a protein at the atomic level. Analy ⁇ i ⁇ require ⁇ the production of crystals and crystal production requires a pure concentrated product. Further, complexes of a protein of interest together with a ⁇ econd interacting molecule provide ⁇ information on the conformational changes occurring within a protein in response to that second molecule. X-ray cry ⁇ tallography of a protein with it ⁇ ⁇ ubstrate, an antibody or a drug can provide information for rational drug design.
  • An X-ray diffraction pattern taken from a crystal looks like an array of ⁇ pot ⁇ of varying intensities. Each spot is related to one of the Fourier coefficients of the electron density pattern in the crystal.
  • the electron density in the cry ⁇ tal can be recon ⁇ tructed if a sufficient number of diffraction spots can be measured and the relative phase angles of the Fourier coefficients can be determined.
  • a crystallized protein u ⁇ ed in the practice of certain aspects of the present invention should be of sufficient quality to obtain these measurements.
  • the spot ⁇ of varying inten ⁇ ity in the diffraction pattern decay over time. It i ⁇ quite difficult to work with diffraction patterns with half lives of less than 10 hours. However, it is possible to work with diffraction patterns having half lives as short as about 15 minutes to 3 hours, depending on the amount of structural data desired to be obtained.
  • phase angles uses isomorphic replacement to insert atoms into defined positions in the crystal for diffraction data measurement. These angles provide information that permit the production of an electron density map. The map is then used to build an atomic model from which three-dimensional coordinates are measured that define the structure of the cry ⁇ tallized molecule.
  • crystal ⁇ of the protein/receptor combination may be u ⁇ ed to gather information on the conformation of the promotor in it ⁇ inhibited bound form.
  • a three-dimensional structure can be obtained from the electron density data using a computer program such as TOM/FRODO.
  • a computer program such as X-PLOR, can be used to improve the accuracy of the initial three- dimensional structure.
  • computer programs available for analyzing X-ray crystallographic data. Those of ordinary skill in the art will recognize that many other such computer programs providing similar functions could also be used. From this data, the points of contact are identified both within the active site and the surrounding region. Invariant amino acids and consensu ⁇ recognition ⁇ equence ⁇ are identified. The data i ⁇ further analyzed against available chemical data such a ⁇ NMR, CD, SANS data and other data re ⁇ ulting from chemical procedures. This chemical data can provide additional information for the structural model.
  • the coordinates of the amino acids residing in and around the active site that may be gleaned from conducting X-ray crystallography on the variou ⁇ fragment ⁇ containing the RERMS ⁇ equence provide a template that may be u ⁇ eful in exploring way ⁇ to enhance interaction with receptor ⁇ .
  • MOLECULE TYPE peptide
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • FRAGMENT TYPE internal
  • MOLECULE TYPE CDNA to mRNA
  • HYPOTHETICAL NO
  • ANTI-SENSE YES
  • SEQUENCE DESCRIPTION SEQ ID NO:3:
  • MOLECULE TYPE CDNA to mRNA
  • HYPOTHETICAL NO
  • ANTI-SENSE YES
  • MOLECULE TYPE peptide
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • FRAGMENT TYPE internal
  • MOLECULE TYPE peptide
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • FRAGMENT TYPE internal
  • MOLECULE TYPE peptide
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • FRAGMENT TYPE internal

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biochemistry (AREA)
  • Biomedical Technology (AREA)
  • Biophysics (AREA)
  • Epidemiology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Genetics & Genomics (AREA)
  • Immunology (AREA)
  • Neurology (AREA)
  • Zoology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Urology & Nephrology (AREA)
  • Hematology (AREA)
  • Cell Biology (AREA)
  • Analytical Chemistry (AREA)
  • Toxicology (AREA)
  • Microbiology (AREA)
  • Neurosurgery (AREA)
  • Food Science & Technology (AREA)
  • Physics & Mathematics (AREA)
  • Biotechnology (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Marine Sciences & Fisheries (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Peptides Or Proteins (AREA)

Abstract

Un peptide isolé plus petit que la protéine naturelle précurseur d'amyloïde (PPA) garde au moins en partie l'effet promoteur de la croissance neuronale de la PPA. Ce peptide comprend au moins cinq résidus successifs d'acides aminés dont les chaînes latérales ont des polarités correspondantes aux polarités des chaînes latérales de la séquence RERMS. L'invention concerne également des composés non peptidiques correspondants et leurs procédés de production. En outre, l'invention concerne des procédés de traitement de maladies neurologiques au moyen de ces peptides et non-peptides.
PCT/US1992/009070 1992-10-23 1992-10-23 Substances ayant l'effet promoteur de croissance de la proteine precurseur d'amyloïde Ceased WO1994009808A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
PCT/US1992/009070 WO1994009808A1 (fr) 1992-10-23 1992-10-23 Substances ayant l'effet promoteur de croissance de la proteine precurseur d'amyloïde
AU28951/92A AU2895192A (en) 1992-10-23 1992-10-23 Substances having the growth-promoting effect of amyloid precursor protein

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/US1992/009070 WO1994009808A1 (fr) 1992-10-23 1992-10-23 Substances ayant l'effet promoteur de croissance de la proteine precurseur d'amyloïde

Publications (1)

Publication Number Publication Date
WO1994009808A1 true WO1994009808A1 (fr) 1994-05-11

Family

ID=22231472

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1992/009070 Ceased WO1994009808A1 (fr) 1992-10-23 1992-10-23 Substances ayant l'effet promoteur de croissance de la proteine precurseur d'amyloïde

Country Status (2)

Country Link
AU (1) AU2895192A (fr)
WO (1) WO1994009808A1 (fr)

Cited By (20)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000018801A3 (fr) * 1998-09-29 2000-07-13 Lars Christian B Roenn Composes se liant aux n-cam
WO2002083729A3 (fr) * 2001-04-18 2003-07-31 Univ Open Polypeptides, leurs derives et leurs utilisations
CN100447153C (zh) * 2000-04-11 2008-12-31 首都医科大学宣武医院 一种新的11肽及其制备方法和用途
US7491702B2 (en) 2001-04-18 2009-02-17 The Open University Polypeptides related to amyloid precursor protein, pharmaceutical compositions thereof, and methods of treatment using the same
US7592304B2 (en) 1999-10-01 2009-09-22 Dmi Life Sciences, Inc. Metal-binding compounds and uses therefor
US7622446B2 (en) 2001-04-18 2009-11-24 The Open University Polypeptides, derivatives and uses thereof
US7632803B2 (en) 1999-10-01 2009-12-15 Dmi Life Sciences, Inc. Metal-binding compounds and uses therefor
US20150283078A1 (en) * 2012-11-19 2015-10-08 Technion Research & Development Foundation Limited Liposomes for in-vivo delivery
US10076384B2 (en) 2013-03-08 2018-09-18 Symple Surgical, Inc. Balloon catheter apparatus with microwave emitter
US10126216B2 (en) 2011-02-17 2018-11-13 Ventana Medical Systems, Inc. Method for tissue sample fixation
US10267769B2 (en) 2010-03-04 2019-04-23 Ventana Medical Systems, Inc. Processing system for processing specimens using acoustic energy
US10531655B2 (en) 2011-12-02 2020-01-14 The Regents Of The University Of California Reperfusion protection solution and uses thereof
US10539487B2 (en) 2010-03-04 2020-01-21 Ventana Medical Systems, Inc. Systems and methods for monitoring tissue sample processing
US10632105B2 (en) * 2013-07-01 2020-04-28 École Polytechnique Fédérale De Lausanne (Epfl) Pharmacological stimulation to facilitate and restore standing and walking functions in spinal cord motor disorders
US11672983B2 (en) 2018-11-13 2023-06-13 Onward Medical N.V. Sensor in clothing of limbs or footwear
US11957910B2 (en) 2011-01-03 2024-04-16 California Institute Of Technology High density epidural stimulation for facilitation of locomotion, posture, voluntary movement, and recovery of autonomic, sexual, vasomotor, and cognitive function after neurological injury
US12023492B2 (en) 2011-11-11 2024-07-02 The Regents Of The University Of California Non invasive neuromodulation device for enabling recovery of motor, sensory, autonomic, sexual, vasomotor and cognitive function
US12357828B2 (en) 2017-12-05 2025-07-15 Ecole Polytechnique Federale De Lausanne (Epfl) System for planning and/or providing neuromodulation
US12415079B2 (en) 2019-11-27 2025-09-16 Onward Medical N.V. Neuromodulation system
US12613624B2 (en) 2019-11-19 2026-04-28 Onward Medical N.V. Planning and/or control system for a neuromodulation system

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4963485A (en) * 1985-01-25 1990-10-16 Oncogen Brain derivable polypeptide factors and antibodies thereto
US5137873A (en) * 1990-07-27 1992-08-11 The Children's Medical Center Corporation Substance p and tachykinin agonists for treatment of alzheimer's disease
US5164295A (en) * 1991-03-06 1992-11-17 The Upjohn Company Method for identifying amyloid protein-extracellular matrix protein affinity altering compounds

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4963485A (en) * 1985-01-25 1990-10-16 Oncogen Brain derivable polypeptide factors and antibodies thereto
US5137873A (en) * 1990-07-27 1992-08-11 The Children's Medical Center Corporation Substance p and tachykinin agonists for treatment of alzheimer's disease
US5164295A (en) * 1991-03-06 1992-11-17 The Upjohn Company Method for identifying amyloid protein-extracellular matrix protein affinity altering compounds

Cited By (29)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000018801A3 (fr) * 1998-09-29 2000-07-13 Lars Christian B Roenn Composes se liant aux n-cam
US7592304B2 (en) 1999-10-01 2009-09-22 Dmi Life Sciences, Inc. Metal-binding compounds and uses therefor
US7632803B2 (en) 1999-10-01 2009-12-15 Dmi Life Sciences, Inc. Metal-binding compounds and uses therefor
CN100447153C (zh) * 2000-04-11 2008-12-31 首都医科大学宣武医院 一种新的11肽及其制备方法和用途
CN100569797C (zh) * 2000-04-11 2009-12-16 首都医科大学宣武医院 一种十一肽及其用途
US7491702B2 (en) 2001-04-18 2009-02-17 The Open University Polypeptides related to amyloid precursor protein, pharmaceutical compositions thereof, and methods of treatment using the same
GB2391548B (en) * 2001-04-18 2005-11-30 Univ Open Polypeptides and derivatives thereof related to amyloid precursor protein (APP)
US7622446B2 (en) 2001-04-18 2009-11-24 The Open University Polypeptides, derivatives and uses thereof
GB2391548A (en) * 2001-04-18 2004-02-11 Univ Open Polypeptides derived from amyloid precursor peptide (APP) and their uses
EP2228388A1 (fr) 2001-04-18 2010-09-15 The Open University Polypeptides protégés Arg-Glu-Arg et leurs utilisations
EP2228387A2 (fr) 2001-04-18 2010-09-15 The Open University Polypeptides dérivés de l'APP (Protéine Précurseur Amyloide), et leurs utilisations
EP2316849A2 (fr) 2001-04-18 2011-05-04 The Open University Polypeptides comprenant RER, dérivés et leurs utilisations
WO2002083729A3 (fr) * 2001-04-18 2003-07-31 Univ Open Polypeptides, leurs derives et leurs utilisations
US10267769B2 (en) 2010-03-04 2019-04-23 Ventana Medical Systems, Inc. Processing system for processing specimens using acoustic energy
US10539487B2 (en) 2010-03-04 2020-01-21 Ventana Medical Systems, Inc. Systems and methods for monitoring tissue sample processing
US11957910B2 (en) 2011-01-03 2024-04-16 California Institute Of Technology High density epidural stimulation for facilitation of locomotion, posture, voluntary movement, and recovery of autonomic, sexual, vasomotor, and cognitive function after neurological injury
US11624684B2 (en) 2011-02-17 2023-04-11 Ventana Medical Systems, Inc. Method for tissue sample fixation
US10126216B2 (en) 2011-02-17 2018-11-13 Ventana Medical Systems, Inc. Method for tissue sample fixation
US12023492B2 (en) 2011-11-11 2024-07-02 The Regents Of The University Of California Non invasive neuromodulation device for enabling recovery of motor, sensory, autonomic, sexual, vasomotor and cognitive function
US12226631B2 (en) 2011-11-11 2025-02-18 The Regents Of The University Of California Non invasive neuromodulation device for enabling recovery of motor, sensory, autonomic, sexual, vasomotor and cognitive function
US10531655B2 (en) 2011-12-02 2020-01-14 The Regents Of The University Of California Reperfusion protection solution and uses thereof
US9655848B2 (en) * 2012-11-19 2017-05-23 Technion Research & Development Foundation Limited Liposomes for in-vivo delivery
US20150283078A1 (en) * 2012-11-19 2015-10-08 Technion Research & Development Foundation Limited Liposomes for in-vivo delivery
US10076384B2 (en) 2013-03-08 2018-09-18 Symple Surgical, Inc. Balloon catheter apparatus with microwave emitter
US10632105B2 (en) * 2013-07-01 2020-04-28 École Polytechnique Fédérale De Lausanne (Epfl) Pharmacological stimulation to facilitate and restore standing and walking functions in spinal cord motor disorders
US12357828B2 (en) 2017-12-05 2025-07-15 Ecole Polytechnique Federale De Lausanne (Epfl) System for planning and/or providing neuromodulation
US11672983B2 (en) 2018-11-13 2023-06-13 Onward Medical N.V. Sensor in clothing of limbs or footwear
US12613624B2 (en) 2019-11-19 2026-04-28 Onward Medical N.V. Planning and/or control system for a neuromodulation system
US12415079B2 (en) 2019-11-27 2025-09-16 Onward Medical N.V. Neuromodulation system

Also Published As

Publication number Publication date
AU2895192A (en) 1994-05-24

Similar Documents

Publication Publication Date Title
EP0890105B1 (fr) Méthods pour le diagnostique, pour la production des médicaments et pour le criblage de substances relatives à la maladie d'Alzheimer et des peptides derivés de l'amyloid beta
WO1994009808A1 (fr) Substances ayant l'effet promoteur de croissance de la proteine precurseur d'amyloïde
EP0638172B1 (fr) Procedes et composition de controle du traitement cellulaire de la proteine precurseur beta-amyloide
DE69333144T2 (de) Verfahren zur Identifizierung von Hemmstoffe der Produktion des beta-Amyloidpeptids
US5605811A (en) Methods and compositions for monitoring cellular processing of beta-amyloid precursor protein
DE69535462T2 (de) Zusammensetzung und Verfahren zur Inhibition der Filamentbildung des Amyloid-Betaproteins und seiner Neutotoxizität
DE69433139T2 (de) Verfahren zur bestimmung von inhibitoren der bildung von beta-amyloidpeptid
KR20080089596A (ko) 치료적 특성을 갖는 베타 1-42 특이적인 단일클론성 항체
WO1995006407A1 (fr) Nouveau composant amyloide lie a la maladie d'alzheimer et procedes d'utilisation
AU681434B2 (en) Methods and compositions for binding TAU and MAP2c proteins
Kisilevsky et al. Pathogenesis of amyloidosis
US5811243A (en) Methods and compositions for binding tau and MAP2c proteins
WO1995026363A1 (fr) Neurotrophine-6: un nouveau membre de la famille des neurotrophines
AU2008200489B2 (en) Methods and compositions for the detection of soluble beta-amyloid peptide
US20030232374A1 (en) Compositions and methods relating to detrusor estrogen-regulated protein (DERP)
AU2004203875A1 (en) Methods and compositions for the detection of soluble beta-amyloid peptide

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AU CA JP KZ US

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): AT BE CH DE DK ES FR GB GR IE IT LU MC NL SE

DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
121 Ep: the epo has been informed by wipo that ep was designated in this application
122 Ep: pct application non-entry in european phase
NENP Non-entry into the national phase

Ref country code: CA