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
  • G01N33/6872—Intracellular protein regulatory factors and their receptors, e.g. including ion channels
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
  • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
  • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
  • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
  • G01N2333/70571—Assays involving receptors, cell surface antigens or cell surface determinants for neuromediators, e.g. serotonin receptor, dopamine receptor
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2500/00—Screening for compounds of potential therapeutic value
  • G01N2500/04—Screening involving studying the effect of compounds C directly on molecule A (e.g. C are potential ligands for a receptor A, or potential substrates for an enzyme A)

Definitions

• the invention relates to the field of neural transmission and drug discovery.
• the conditions surrounding the transmission of signals through neuronal networks clearly affects a variety of physiological responses, including perception of pain, learning, memory, and the like. Modulation of the level of neural transmission and the condition of the presynaptic environment have profound physiological effects, primarily within the nervous system.
• the primary calcium ion channels that effect neural transmission are these N and P/Q type channels.
• P/Q type channels have been implicated mostly in the presynaptic terminals of the central nervous system (CNS) while the N type channels appear to dominate in the pe ⁇ pheral nervous system.
• P/Q type channels are particularly important in CNS functions such as memory and pain
• ⁇ ⁇ subunit contains a sequence which binds to the known protein Homer which is desc ⁇ bed in articles by Xiao, B , et al , Cur Opinion in ⁇ eurob ⁇ ol (2000) 10 370-374 and by Tu, ] C , et al euron (1998) 21 717-726
• the disclosures of these articles are incorporated herein by reference
• the Homer protein binds to a multiplicity of targets which are important in signaling and neurotransmission
• a consensus sequence which is prohne ⁇ ch is also desc ⁇ bed
• the invention resides in the identification of a peptide region specific to the P Q calcium ion channel that is responsible for the casca ⁇ e of e ents that results in expression of the gene encoding syntaxin- 1 A
• use of this peptide in screening assays permits identification of compounds that can be used to regulate the levels of syntaxin- 1 A available in the presynaptic region and thus modulate such functions as learning, memory and pain
• P/Q ion channel specifically effects the expression of the gene encoding syntaxin in model cell systems and in neuronal cells per se It has now been found that a specific 4-am ⁇ no acid sequence approximately 200 amino acids from the C terminus of the P/Q calcium ion channel is the site for interaction of this channel with Homer, a protein known to affect intracellular calcium ion stores, and this interaction is essential for the ability of the P Q calcium ion channel to effect the expression of the syntaxin- 1 A encoding gene
• the invention is directed to a method to identify compounds that affect central nervous system function, such as learning and memory, hich method comp ⁇ ses contacting a candidate compound with a peptide that comp ⁇ ses the binding site for Homer which resides proximal to the carboxy terminus of the P/Q calcium ion channel and determining whether said compound binds to said
• the invention is directed to a peptide comprising the sequence of the binding site flanked by additional amino acids, typically those which flank the binding site in the native ion channel, and to antibiotics that are immunospecific for this region.
• the invention is directed to the compounds so identified and to methods to modulate CNS function using these compounds.
• syntaxin- 1 A a presynaptic protein that plays a central role in mediating vesicle docking, fusion and neurotransmitter release.
• syntaxin- 1 A a presynaptic protein that plays a central role in mediating vesicle docking, fusion and neurotransmitter release.
• the initial calcium ion signal is amplified through calcium ion from intracellular stores and acts via phosphorylation that is dependent on a number of cofactors including CaMK ⁇ /IV, PICA., and MAPKK.
• syntaxin- 1 A interacts with P/Q type calcium ion channels to decrease channel availability, the expression of the gene encoding syntaxin- 1 A is regulated by an activity-dependent feedback pathway.
• the specific site required for binding is the four amino acid sequence PLMF.
• additional amino acid sequence at one or both of the N and C terminus of this tetrapeptide.
• Preferred embodiments for such additional amino acid sequence are the sequences present in the native ion channels. Typically, sequences extending 20 amino acids upstream and/or downstream, more preferably 15 amino acids upstream and/or downstream, or even 10 or 5 or 2 amino acids of the sequences upstream and/or downstream of the required tetrapeptide are employed.
• sequence containing the tetrapeptide with the tetrapeptide itself in bolded and underlined types is as follows:
• This sequence is derived from one allele encoding a human P/Q type calcium channel. It is understood that similar sequences are included in the corresponding P/Q channels of other mammalian species, and peptides de ⁇ ved from these species as well as from allelic variants of the sequence shown above may be used in the method of the invention. In general, it is preferred to utilize peptides containing sequence derived from the above-shown sequence which are at least 90% homologous, preferably 95% homologous, more preferably 97% homologous and more preferably
• syntaxin- 1 A was not endogenously expressed in HEK293 cells while the presence of the syntaxin- 1 A protein was detected in HEK293 cells transfected with the d ⁇ A P/Q type calcium channel; mRNA corresponding to syntaxin- 1 A was also detected, but inhibited when the cells were incubated in actinomycin D
• Syntax ⁇ n-1 A was the only SNARE protein produced in these cells; syntaxin IB, SNAP-25, synaptophysin, VAMP, and synaptotagmin were not detected There as no evidence for production of syntaxin- 1 A in HEK293 cells transfected with calcium ion channel ⁇ 2 ⁇ or ⁇ , B subunits. Cells transfected with ⁇ B
• Syntaxin- 1 A expression in ⁇ A transfected cells could be inhibited with compounds known to block tyrosine kinase, calmodulin activity or CaMK ⁇ /IV or PKA activities.
• Activation of protein kinase C did not induce expression.
• a portion of the transduction pathway may also involve the transc ⁇ ption factor CREB, as it was determined that the level of phosphorylated CREB is up-regulated in the transfected HEK cells.
• P/Q type selective calcium influx induces expression of syntaxin- 1 A but not of other SNARE proteins involved in vesicle fusion and neurotransmitter release. It appears to be under tight spatial and temporal calcium dependent control and is apparently mediated by an association with intracellular calcium stores.

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• Peptides Or Proteins (AREA)
• Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
• Investigating Or Analysing Biological Materials (AREA)
• Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
• Acyclic And Carbocyclic Compounds In Medicinal Compositions (AREA)
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• 2000
  • 2000-10-13 CA CA002385907A patent/CA2385907A1/en not_active Abandoned
  • 2000-10-13 ES ES00969145T patent/ES2253263T3/es not_active Expired - Lifetime
  • 2000-10-13 AU AU78967/00A patent/AU7896700A/en not_active Abandoned
  • 2000-10-13 JP JP2001530589A patent/JP2003511087A/ja active Pending
  • 2000-10-13 WO PCT/CA2000/001233 patent/WO2001027630A2/en not_active Ceased
  • 2000-10-13 US US09/688,295 patent/US6531288B1/en not_active Expired - Fee Related
  • 2000-10-13 EP EP00969145A patent/EP1230551B1/en not_active Expired - Lifetime
  • 2000-10-13 AT AT00969145T patent/ATE312350T1/de not_active IP Right Cessation
  • 2000-10-13 DE DE60024664T patent/DE60024664T2/de not_active Expired - Fee Related

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