WO2009005783A1 - Peptides, compositions et procédés pour réduire l'apoptose médiée par les bêta-amyloïdes - Google Patents

Peptides, compositions et procédés pour réduire l'apoptose médiée par les bêta-amyloïdes Download PDF

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WO2009005783A1
WO2009005783A1 PCT/US2008/008147 US2008008147W WO2009005783A1 WO 2009005783 A1 WO2009005783 A1 WO 2009005783A1 US 2008008147 W US2008008147 W US 2008008147W WO 2009005783 A1 WO2009005783 A1 WO 2009005783A1
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seq
peptide
peptides
amino acid
acid sequence
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PCT/US2008/008147
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Thomas J. Nelson
Daniel L. Alkon
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Blanchette Rockefeller Neurosciences Institute
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids

Definitions

  • the present invention provides peptides, antibodies and anti-idiotypic antibodies that reduce A ⁇ (39-42)-mediated apoptosis, as compared to untreated neuronal cells.
  • AD Alzheimer's Disease
  • a ⁇ beta-amyloid
  • the extracellular A ⁇ plaque deposits are composed of a proteinacious core of insoluble aggregated A ⁇ peptides, from 39-42 amino acids in length A ⁇ (l-42).
  • the presence of these aggregates has led to the foundation of the amyloid hypothesis. This hypothesis postulates that A ⁇ (l-42) is one of the principal causative factors of neuronal death in the brains of Alzheimer's patients.
  • Beta-amyloid is a toxic peptide produced by cleavage of amyloid precursor protein
  • a ⁇ peptide produces apoptosis in neurons but the mechanism by which A ⁇ exerts its toxic effect is unknown.
  • Application of A ⁇ to cultured neuronal cells at micromolar concentrations causes apoptosis, and lower concentrations cause upregulation of apoptosis markers caspase 3 and annexin (White, et al., Neurobiol Dis. 2001 ; 8, 299-316).
  • Biochemical effects of A ⁇ include activation of calcium channels, production of free radicals, excitotoxicity through activation of NMDA receptors, and glutamate accumulation leading to increased Ca2+ levels.
  • Intracerebroventricular injection of A ⁇ produces impairment of spatial memory and non-spatial long term memory, reduction of protein kinase C (PKC) activity, induction of apoptosis, and activation of astrocytes and microglia to release excessive amounts of inflammatory cytokines.
  • Transgenic animals expressing human A ⁇ exhibit many of the pathologies of Alzheimer's disease (AD), including cognitive deficits, age-related formation of amyloid plaques, activation of astrocytes and microglial cells, vascular amyloid pathology, degeneration of cholinergic nerve terminals, and reduced lifespan.
  • AD Alzheimer's disease
  • transgenic mice expressing normal human APP do not exhibit the neurofibrillary tangles and significant neuronal loss characteristic of AD (German, et al., Rev Neurosci, 2004; 15, 353-369).
  • Down syndrome trisomy 21 patients invariably develop (in their third or fourth decade) cerebral amyloid plaques and neurofibrillary tangles, the characteristic lesions of Alzheimer disease (AD).
  • AD Alzheimer disease
  • a ⁇ (l-42) is the earliest form of this protein deposited in Down syndrome brains, and may be seen in subjects as young as 12 years of age, and that soluble A ⁇ can be detected in the brains of Down syndrome subjects as early as 21 gestational weeks of age, well preceding the formation of A ⁇ plaques (Gyure et al., Archives of Pathology and Laboratory Medicine, 2000; 125, 489-492).
  • Congophilic angiopathy also referred to as amyloid angiopathy
  • a ⁇ deposits in the walls of the leptomeninges and superficial cerebral cortical blood vessels of the brain.
  • Amyloid deposition predisposes these blood vessel to failure, increasing the risk of a hemorrhagic stroke.
  • brain hemorrhages are more common in people who suffer from Alzheimer's, however they can also occur in those who have no history of dementia.
  • a ⁇ formation of A ⁇ from APP is dependent on the intracellular transport system. APP is transported from the ER and Golgi to the cell surface membrane, where it may be cleaved into the non- amyloidogenic peptide by the enzyme ⁇ -secretase. PKC-activated ⁇ -secretases also reside in the trans- Golgi network (TON) (Skovronsky, et al., J. Biol. Chem., 2000; 275, 2568-2575), which is a major site for ⁇ -secretase activity. Uncleaved APP is then internalized into endocytic compartments, where it is cleaved by ⁇ - and ⁇ -secretase to produce A ⁇ . A ⁇ can also be produced in the Golgi and ER compartments.
  • TON trans- Golgi network
  • APP is an integral membrane protein with high affinity for copper (White, et al., J Neurosci, 2002; 19, 9170-9179 and Barnham, et al., J Biol Chem, 2003; 278, 17401-17407). It has been suggested that APP is involved in neurodevelopment (Grilli, et al., Funct Neurol, 2003; 18, 145-148) and is essential for neuronal growth. Mutant mice in which APP has been knocked out develop reactive gliosis, weight loss, cognitive defects, and reduced levels of presynaptic marker proteins (Dawson, et al., Neuroscience, 1999; 90, 1-13), indicating generalized CNS pathology.
  • APP Downregulation of APP inhibits neurite outgrowth (Allinquant, et al., J. Cell Biol., 1995; 128, 919-927) and anti-APP antibodies block memory formation in chicks (Mileusnic, et al., Eur J Neurosci., 2000; 12, 4487-4495.).
  • APP After being transported along nerve fibers, APP participates in synaptogenesis (Moya, et al., Dev. Biol., 1994; 161 , 597-603) and cell adhesion (Small, et al., J Alzheimers Dis., 1999; 1, 275-285).
  • synaptogenesis Moya, et al., Dev. Biol., 1994; 161 , 597-603
  • cell adhesion Small, et al., J Alzheimers Dis., 1999; 1, 275-285.
  • APP may play an important role in normal synaptic plasticity and neuronal growth.
  • a ⁇ peptide is also found in CSF and blood plasma of normal patients (Seubert, et al., Nature, 1992; 359, 325-327).
  • the production and secretion of A ⁇ is regulated by neuronal activity (Kamenetz, et al., Neuron, 2003; 37, 925-937).
  • Kamenetz et al. found that APP reversibly depresses synaptic transmission by a mechanism mediated by activation of APP cleavage by NMDA receptors. This suggests that A ⁇ is normally produced by neurons and has one or more functions in normal cells.
  • sortilin receptor SorLA also interacts with ApoE (45) and regulates A ⁇ production and APP traffic in endocytic compartments (46). Sortilin is also reported to be a substrate for gamma-secretase and possibly PKC-mediated alpha-secretase (Nyborg, et al., MoI Neurodegener, 2006; 1 , 1-1 1). Its apparent role is to target proteins in the Golgi for transport to late endosomes.
  • the 14-3-3 protein which has many similarities to the Parkinson's disease- associated protein ⁇ -synuclein (Ostrerova, et al., J Neurosci., 1991 ; 19, 5782-5791), is found in neurofibrillary tangles (Layfield, et al., Neurosci Lett, 1996; 209, 57-60), binds to tau, and is involved in phosphorylation of tau by GSK-3 ⁇ (Li, et al., Neurosci Lett, 2007; 414, 203-208 and Hashiguchi, et al., J Biol Chem, 2004; 275, 25247-25254).
  • GSK-3f3 which is the principal enzyme involved in phosphorylating tau, is regulated by 14-3-3 (Yuan, et al., J Biol Chem, 2004; 279, 26105-261 14). Increases in 14-3-3 have been reported in patients with Alzheimer's disease (Fountoulakis, et al., J Neural Transm Suppl, 1999; 57, 323-335). Phosphorylation of Ser-9 in GSK-3P promotes binding of GSK-3p to 14-3-3.
  • 14-3-3 zeta was also one of a small number of proteins found to be significantly oxidized after intracerebral injection of A ⁇ 1 -42 (Boyd- Kimball, et al., Neuroscience; 2005; 132, 313-324). Thus, binding of A ⁇ to 14-3-3 could be a missing link that connects two important pathways in AD (A ⁇ oligomerization and neurofibrillary tangles).
  • Orner et al also recently used phage display to identify proteins that might mediate aggregation of A ⁇ (Orner, et al., J Am Chem Soc, 20; 128, 1 1882-1 1889.).
  • the predominant peptide motif found by of Orner et al. was a fragment identical to a region of A ⁇ itself (QKLVFF; SEQ ID NO: 29), containing the ⁇ -secretase cleavage site and the "hydrophobic patch" (Fig. 5), suggesting that this region is critical for A ⁇ aggregation.
  • CCA collagenous Alzheimer amyloid plaque component
  • ⁇ l isoform 1 a proteolytic form of collagen type XXV, ⁇ l isoform 1 ; ⁇ 2-macroglobulin, a protease inhibitor that is released in response to inflammatory stimuli (Hughes, et al., Proc Natl Acad Sci USA, 1998; 95, 3275-3280 and Narita, et al., J Neurochem, 1997; 69, 195-204); tau (Prez, et al., J Alzheimers Dis; 2004; 6,461-467); the collagen-like domain of complement CIq A chain (Jiang, et al., J Immunol, 1994; 152, 5050-5059); the p75 neurotrophin
  • LRP low-density lipoprotein receptor related protein
  • a ⁇ interacts with tubulin, CNPase, and myelin basic protein, which was found by
  • Verdier et al. who studied synaptosomal proteins that co-precipitated with fibrillar A ⁇ . Differences between their results and ours may he attributed to their focus on membrane- extracted proteins from the synapse. Since A ⁇ is created in the endoplasmic reticulum (A ⁇ 1-42), trans-Golgi network (A ⁇ 1-40), and endocytic compartments (A ⁇ 1 -40) (Greenfield, et al., Proc Natl Acad Sci USA, 1999; 96, 742-747 and Soriano, et al., J Biol. Chem, 1999; 274, 32295- 32300), the results of Verdier et al, may relate more specifically to A ⁇ 's possible functions in synaptic function or synaptic vesicle endocytosis.
  • Verdier et al. indicate that some form of phosphodiesterase 3 or CNPase interacts with A ⁇ .
  • a ⁇ had only a minor effect on PDE activity, and co- precipitation experiments from brain cytosol or detergent extracts showed little evidence of a direct interaction. Therefore, it is possible that the interaction between A ⁇ and PDE is indirect.
  • Phorbol ester-induced phosphorylation of PDE 3A promotes binding to 14-3-3 (Rubio, P. Biochem J, 2005; 392, 163- 172).
  • Phosphorylation of PDE 3B by A-kinase also promotes binding to 14-3-3 (Palmer, et al., J Biol Chem., 2007; 282, 941 1-9419).
  • the interaction of A ⁇ with phosphodiesterase and CNPase may be mediated by 14-3-3. Further investigation of the interactions between A ⁇ and 14-3-3 may shed additional light on the possible involvement of PDE and CNPase.
  • CRP C-reactive protein
  • a member of the pentraxin family is an acute-phase protein normally found in plasma.
  • CRP immunoreactivity is also detectable in temporal cortex of AD patients (Wood, et al., Brain Res., 1993; 629, 245-252), more specifically in neurofibrillary tangles (Duong, et al., Brain Res, 1997; 749, 152-156 and McGeer, et al., Neurobiol Aging, 2001 ; 22,843-848).
  • CRP mRNA is also detectable in pyramidal neurons, indicating that it is synthesized in the brain, and CRP is upregulated in AD (Yasojima, et al., Brain Res, 2000; 887, 80-89).
  • AD Yasojima, et al., Brain Res, 2000; 887, 80-89.
  • Patients with the pathogenic apolipoprotein APOE4 allele have lower levels of C- reactive protein than normal patients (Haan, et al., Neurobiol Aging, 2007; May 29, 0-0).
  • the question of whether inflammation in AD is a pathogenic event, a response to neurodegeneration, or possibly even a beneficial response, has not been resolved.
  • the possibility of new functions for CRP also cannot be ruled out.
  • Pentraxins such as the related protein neuronal pentraxin 1
  • Pentraxin 1 can mediate neuronal apoptosis following the loss of neuronal activity (DeGregorio-Rocasolano, et al., J Biol Chem, 2001 ; 276, 796-803).
  • Pentraxin I is increased in dystrophic neurites in patients with sporadic late-onset AD (Abad, et al., J Neurosci., 2006; 26, 12735-12747).
  • a ⁇ also binds to the ER-Golgi intermediate compartment (ERGIC) marker protein
  • ERGIC-53 which is involved in the calcium-dependent transport of glycoproteins such as APP from the ER to the Golgi intermediate compartment (Itin, et al., MoI Biol Cell, 1996; 7, 483-493), where presenilin is located (Culvenor, et al., J Neurosci Res, 1997; 49, 719-731).
  • ERGIC- 53 like sortilin, may serve a transport function.
  • Nicastrin a glycoprotein component of the ⁇ - secretase complex, interacts with ERGIC-53 (Morais, et al., Biochem Biophys Acta, 2006; 1762, 802-810).
  • ERG(C2 originally named PTX I
  • PTX I is a similar transport protein originally found in prostate (Kwok et al., DNA Cell Biol, 2001 ; 20, 349-357) and may also serve to transport APP or A ⁇ .
  • Apolipoprotein B Apolipoprotein B (ApoB), like ApoE, is a component of low- density lipoproteins, and is a suspected factor in atherosclerosis. ApoE4 is a genetic predisposing factor in AD.
  • Serum ApoB levels are increased in AD (Caramelli, et al., Acta Neural Scand., 1999; 100, 61 -63 and Kuo, et al., Biochem Biophys Res Commun, 1998; 252, 71 1 -715) and although predominantly a serum protein, ApoB is also found in hippocampus, where it is associated with hippocampal amyloid deposits and neurofibrillary tangles (Namba, et al., Neurosci Lett, 1993; 134, 264-266).
  • Overexpression of ApoB in mice increases APP expression in mice fed a high-cholesterol diet (Bjelik, et al., Neurochem. Int, 2006; 49, 393-400).
  • Apolipoproteins function to transport cholesterol to the cell.
  • ApoB, ApoE, steroid reductase, and sortilin all of which are involved in some way with low-density lipoproteins and cholesterol transport or metabolism, is further evidence of involvement of A ⁇ with cholesterol uptake or metabolism.
  • There is considerable evidence for a role of cholesterol or its metabolites in AD (Evans, et al., Neurology, 2004; 62, 1869-1871 ; Puglielli, et al., Nat.
  • P2X2 Purinergic receptors are upregulated in AD. Recent studies have shown that caffeine and adenosine receptor antagonists prevent A ⁇ -induced cognitive deficits in mice (Dall'Igna, et al., Exp Neural, 2007; 203, 241 -245) and reduces A ⁇ production (Arendash, et al., Neuroscience, 2006; 142, 941 -952). P2X2 has been shown to interact with Fe65 (Masin, et al., J Biol Chem, 2006; 281 , 4100-4108), an adaptor protein for APP that associates with tau in vivo (Barbato, et al., Neurobiol Dis, 2005; 18, 399-408).
  • the present invention provides isolated peptides having amino acid sequences
  • VSVGMLWC (SEQ ID NO: 4); SVLDRQRC (SEQ ID NO: 5); LGSYKPSC (SEQ ID NO: 6); NDRGLLAC (SEQ ID NO: 11); and YQDSAKTC (SEQ ID NO: 10).
  • the present invention also provides nucleic acids, both RNA and DNA, encoding the peptides of SEQ ID NO: 4, 5, 6, 10 and 1 1.
  • the present invention also discloses cloning and expression vectors comprising the nucleic acids of the present invention operably linked to a promoter.
  • the present invention also provides host cells, including prokaryotic and eukaryotic cells, such as nonpathogenic yeast, transformed with the vectors of the present invention.
  • the present invention also provides methods of producing the peptides of SEQ ID NO: 4, 5, 6, 10 and 11 comprising the steps of culturing the transformed host cells and isolating the expressed peptides.
  • compositions comprising an isolated peptides having amino acid sequences VSVGMLWC (SEQ ID NO: 4); SVLDRQRC (SEQ ID NO: 5); LGSYKPSC (SEQ ID NO: 6); NDRGLLAC (SEQ ID NO: 1 1); and YQDSAKTC (SEQ ID NO: 10) in a pharmaceutically acceptable carrier.
  • the present invention also provides a method of reducing A ⁇ (39-42)-mediated apoptosis in a neuronal cell, which method comprises contacting the neuronal cell with an effective amount of at least one of the following peptides: VSVGMLWC (SEQ ID NO: 4); SVLDRQRC (SEQ ID NO: 5); LGSYKPSC (SEQ ID NO: 6); NDRGLLAC (SEQ ID NO: 1 1); or YQDSAKTC (SEQ ID NO: 10), as compared to untreated neuronal cells.
  • FIG. 1 depicts results of co-precipitation experiments of A ⁇ 1-42 and A ⁇ - binding proteins in rabbit brain extracts.
  • Fig. IA shows co-precipitation with C-reactive pre-cursor protein; GDNF receptor; steroid 5 ⁇ -reductase; 14-3-3 ⁇ ; and 14-3-3 ⁇ .
  • Fig. IB shows co-precipitation with ERGlC-52; ERGIC-2; sortilin; P2XP and ApoB.
  • FIG. 2 depicts A ⁇ -binding proteins identified in mice by results of mass spectrometry.
  • FIG. 3A-C shows results of peptides derived from A ⁇ -binding proteins on the activity of purified phosphodiesterase 3B or phosphodiesterase 3B purified from rabbit brain homogenates.
  • Fig. 3A shows total phosphodiesterase activity.
  • Fig. 3B shows the effect on purified phosphodiesterase 3A.
  • Fig. 3C shows effect on purified phosphodiesterase 3B.
  • FIG. 4 shows the effects of peptides derived from A ⁇ 1-42 on apoptosis of cultured neuronal cells.
  • Fig. 4A shows the effects of peptides on pBad.
  • Fig. 4B shows the effects on caspase.
  • Fig. 4C shows the effects on ⁇ -galactosidase, an indicator of cell senescence.
  • FIG. 5 depicts the regions of A ⁇ 1-42 which bind to the A ⁇ -binding proteins and peptides derived from such proteins.
  • a ⁇ was found to interact with apolipoprotein B and the C- reactive protein precursor, the cholesterol transporter proteins sortilin, ERGIC2, and ERGIC53, and the regulatory proteins 14-3-3 epsilon and 14-3-3 gamma.
  • peptides bound to the hydrophobic region (residues 17-21) or to the nearby PKC pseudo-phosphorylation site (residues 26-30) of A ⁇ , regions involved in A ⁇ 's effector action and aggregation. As described in more detail in the Examples, peptides based on these binding regions were effective blockers of A ⁇ toxicity.
  • isolated means that the referenced material is removed from the environment in which it is normally found.
  • an isolated biological material can be free of cellular components, i.e., components of the cells in which the material is found or produced.
  • an isolated peptide is free from other proteins or nucleic acids, or both, in which it is comprised or with which it associates in the cell.
  • An isolated material may be, but need not be, purified.
  • purified refers to material, such as a peptide of the present invention, that has been isolated under conditions that reduce or eliminate unrelated materials, i.e., contaminants.
  • a purified peptide is preferably substantially free of other proteins or nucleic acids with which it is associated in a cell.
  • substantially free is used operationally, in the context of analytical testing of the material.
  • purified material substantially free of contaminants is at least 50% pure; more preferably, at least 90% pure, and more preferably still at least 99% pure. Purity can be evaluated by conventional means, e.g., chromatography, gel electrophoresis, immunoassay, composition analysis, biological assay, and other methods known in the art.
  • ⁇ -amyloidoses or a “disorder associated with A ⁇ ” refers to amyloid diseases which involve the formation, deposition, accumulation and/or persistence of A ⁇ (i.e., beta- amyloid protein), including but not limited to A ⁇ containing 39-43 amino acids in length, but more preferably, A ⁇ 1-40, or A ⁇ 1-42, and mixtures or fragments thereof.
  • a ⁇ i.e., beta- amyloid protein
  • Such disorders include but are not limited to but are not limited to Alzheimer's disease, Down's syndrome, forms of familial amyloidosis, cerebrovascular amyloidosis and cerebral hemorrhage; cystatin C amyloid angiopathy; hereditary cerebral hemorrhage with amyloidosis (Dutch type); hereditary cerebral hemorrhage with amyloidosis (Icelandic type); and inclusion body myositis.
  • patient or "patient population” or “individual in need thereof refer to individual(s) diagnosed as having a ⁇ -amyloidosis, or at risk of developing a ⁇ -amyloidosis, such as but not limited to AD or Down Syndrome. Methods of and criteria for diagnosis of such disorders is described further below.
  • a therapeutic response may be any response that a user (e.g., a clinician) will recognize as an effective response to the therapy.
  • a therapeutic response will generally be an amelioration or inhibition of one or more symptoms of a disease or disorder.
  • a therapeutic response to AD may include amelioration of the foregoing symptoms and surrogate clinical markers of ⁇ -amyloidoses, such as inhibition of neuronal apoptosis or reduction in A ⁇ deposits or aggregates. Exemplary methods for evaluating a therapeutic response also are described further below.
  • pharmaceutically acceptable refers to molecular entities and compositions that are physiologically tolerable and do not typically produce untoward reactions when administered to a human.
  • pharmaceutically acceptable means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.
  • carrier refers to a diluent, adjuvant, excipient, or vehicle with which a therapeutic agent is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils.
  • Water or aqueous solution saline solutions and aqueous dextrose and glycerol solutions are preferably employed as carriers, particularly for injectable solutions.
  • Suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences” by E. W. Martin, 18th Edition, or other editions.
  • antibody is meant to include polyclonal antibodies, monoclonal antibodies
  • mAbs chimeric antibodies, anti-idiotypic (anti-Id) antibodies to antibodies that can be labeled in soluble or bound form, as well as fragments, regions or derivatives thereof, provided by any known technique, such as, but not limited to enzymatic cleavage, peptide synthesis or recombinant techniques.
  • a ⁇ refers to a human A ⁇ polypeptide having an amino acid sequence set forth in SEQ ID NO: 1 and Figure 5.
  • “Function-conservative variants” are those in which a given amino acid residue in a protein has been changed without altering the overall conformation and function of the polypeptide, including, but not limited to, replacement of an amino acid with one having similar properties (such as, for example, polarity, hydrogen bonding potential, acidic, basic, hydrophobic, aromatic, and the like).
  • Amino acids with similar properties are well known in the art. For example, arginine, histidine and lysine are hydrophilic-basic amino acids and may be interchangeable. Similarly, isoleucine, a hydrophobic amino acid, may be replaced with leucine, methionine or valine.
  • a “function-conservative variant” also includes a polypeptide which has at least at least
  • the terms “about” and “approximately” shall generally mean an acceptable degree of error for the quantity measured given the nature or precision of the measurements. Typical, exemplary degrees of error are within 20 percent (%), preferably within 10%, and more preferably within 5% of a given value or range of values. Alternatively, and particularly in biological systems, the terms “about” and “approximately” may mean values that are within an order of magnitude, preferably within 5-fold and more preferably within 2-fold of a given value. Numerical quantities given herein are approximate unless stated otherwise, meaning that the term “about” or “approximately” can be inferred when not expressly stated.
  • the present invention provides novel isolated peptides that competitively bind to A ⁇ , thereby blocking pathological A ⁇ effector functions. Also provided are antibodies raised against such peptides, and anti-idiotypic antibodies raised against the anti-peptide antibodies. Methods of using the peptides and anti-idiotypic antibodies for the treatment or prevention of ⁇ -amyloidoses associated with A ⁇ also are provided.
  • One embodiment of the invention provides novel peptides which bind to amino acid residues 17-21 of human A ⁇ (LVFFA; SEQ ID NO: 2).
  • the peptide comprises the sequence VSVGMLWC (SEQ ID NO:
  • the peptide comprises the sequence SVLDRQRC
  • the peptide comprises the sequence LGSYKPSC (SEQ ID NO: 1]
  • the peptides contain no more than two amino acid substitutions with function-conserved variants, preferably no more than one function-conserved variant.
  • the amino acids of peptide VSVGMLWC can have the following amino acid function-conserved substitutions: i) valine is replaced with other aliphatic, hydrophobic amino acids such as isoleucine or leucine; ii) serine is replaced with other polar, small amino acids such as threonine; iii) glycine is preferably not replaced; iv) methionine is replaced with other hydrophobic amino acids such as leucine, isoleucine or valine; v) leucine is replaced with other aliphatic hydrophobic amino acids such as methionine, isoleucine, or valine; vi) tryptophan is replaced with other aromatic, hydrophobic amino acids such as tyrosine or phenylalanine; and vii) cysteine is replaced with small amino acids but is preferably not replaced.
  • amino acids of peptide SVLDRQRC can have the following amino acid function-conserved substitutions: i) serine is replaced with other polar, small amino acids such as threonine; ii) valine is replaced with other aliphatic, hydrophobic amino acids such as isoleucine or leucine; iii) leucine is replaced with other aliphatic hydrophobic amino acids such as methionine, isoleucine, or valine; iv) aspartic acid is replaced with other negatively charged, polar amino acids such as glutamic acid or asparagine; v) arginine is replaced with other positively charged, polar amino acids such as lysine or glutamine; vi) glutamine is replaced with other polar amino acids such as glutamic acid; and vii) cysteine is preferably not substituted.
  • the amino acids of peptide LGSYKPSC can have the following amino acid function-conserved substitutions: i) leucine is replaced with other aliphatic hydrophobic amino acids such as methionine, isoleucine, or valine; ii) glycine is preferably not substituted; iii) serine is replaced with other polar, small amino acids such as threonine; iv) tyrosine is replaced with other aromatic, more hydrophobic amino acids such as phenylalanine or tryptophan, or sometimes histidine; v) lysine is replaced with other positively charged amino acids such as arginine, glutamic acid, or glutamine; vi) proline is preferably not substituted; vii) serine is replaced with other polar, small amino acids such as threonine; and viii) cysteine is preferably not substituted.
  • the peptides of the present invention may contain chemical modifications to increase physical or chemical stability ex vivo during synthesis or in formulations or in vivo following administration to an individual. Such modifications include capping and pegylation.
  • the peptides are modified, or "capped,” at the N or C termini.
  • Exemplary modifications include acetylation at the N terminus (N-acetylation) and amidation at the C terminus (C-amidation). Such modifications protect the peptide from degradation and make the peptide more closely mimic the charge state of the alpha amino and carboxyl groups in the native protein.
  • modifications include the conjugation of polymers to peptides to improve their stability, solubility, and circulating half-life, and protect peptides from degradation.
  • typical reactive amino acids include lysine, cysteine, histidine, arginine, aspartic acid, glutamic acid, serine, threonine, tyrosine.
  • the N-terminal amino group and the C-terminal carboxylic acid can also be used.
  • Exemplary polymers include linear or branched polyethylene glycols (PEG) and derivatives and other polyvinylalcohols (PVA).
  • PEG polyethylene glycols
  • PVA polyvinylalcohols
  • the anti-peptide antibodies are monoclonal (mAb).
  • the mAbs may be obtained by methods known to those skilled in the art. See, for example Kohler and Milstein, Nature 256:495-497 (1975); U.S. Pat. No.
  • anti-idiotypic antibodies to the anti-peptide antibodies described above. Hypervariable regions of an antibody (AbI) can themselves act as antigens. The antibodies produced in this way are known as anti-idiotype antibodies (Ab2) since they bind to the idiotypic region (binding site) of the first antibody. Such anti-idiotypic antibodies therefore represent an "internal image" of the original antigen, i.e., a structural mimic of the antigen to which the first antibody (AbI) was raised. Thus, anti-idiotypic antibodies raised against antibodies which specifically bind to the A ⁇ -derived peptides of the present invention will structurally mimic the peptides. Accordingly, such anti-idiotypic antibodies and fragments thereof are contemplated for use in the method of the present invention since they will prevent A ⁇ binding to its effector proteins, thereby inhibiting pathologic A ⁇ -mediated effects.
  • the anti-idiotypic antibodies also may be chimeric.
  • Chimeric antibodies are molecules different portions of which are derived from different animal species, such as those having variable region derived from a murine mAb and a human immunoglobulin constant region (Fc), which are primarily used to reduce immunogenicity in application and to increase yields in production, for example, where murine mAbs have higher yields from hybridomas but higher immunogenicity in humans, such that human/murine chimeric mAbs are used.
  • Chimeric antibodies and methods for their production are known in the art (Cabilly et al, Proc. Natl. Acad. Sci. USA. 1984; 81 :3273-3277). Chimeric antibodies containing a human Fc region are referred to as "humanized" antibodies.
  • antibody fragments derived from naturally-occurring anti-idiotypic antibodies. Such fragments will comprise either both heavy and light chains (e.g., Fab fragments), or single heavy or light chains (e.g., light chain dimers), together with their constant region component stretches.
  • the antibody fragment may be a single-chain variable region (scFv), which is a fusion of the variable regions of the heavy and light chains of immunoglobulin, linked together with a short (usually serine, glycine) linker.
  • the Fab or scFv antibody fragments can be generated using phage display.
  • phage display technique gene segments encoding the antigen-binding variable domains of antibodies are fused to genes encoding the coat protein of a bacteriophage. Bacteriophage containing such gene fusions are used to infect bacteria, and the resulting phage particles have coats that express the antibody-like fusion protein, with the antigen-binding domain displayed on the outside of the bacteriophage.
  • a collection of recombinant phage, each displaying a different antigen-binding domain on its surface, is known as a phage display library.
  • Phage expressing antigen-binding domains specific for a particular antigen can be isolated by selecting the phage in the library for binding to that antigen.
  • the phage particles that bind are recovered and used to infect fresh bacteria.
  • Each phage isolated in this way produces a monoclonal antigen-binding particle analogous to a monoclonal antibody.
  • the genes encoding the antigen-binding site, which are unique to each phage can then be recovered from the phage DNA. When the genes encoding antibody fragment are introduced into a suitable host cell line, such as bacteria, the transfected cells can secrete antibody fragments.
  • apoptosis in a neuronal cell comprising the step of contacting the neuronal cell with an effective amount of a composition comprising a combination of one or more of the peptides or antibodies of the present invention and a therapeutic agent selected from the group consisting of Cognex ® (tacrine), Aricept ® (donepezil), Exelon ® (rivastigmine), Reminyl ® (galantamine), and NMDA receptor antagonists such as Namenda ® (memantine).
  • a therapeutic agent selected from the group consisting of Cognex ® (tacrine), Aricept ® (donepezil), Exelon ® (rivastigmine), Reminyl ® (galantamine), and NMDA receptor antagonists such as Namenda ® (memantine).
  • Other suitable therapeutic agents include protease inhibitors (see e.g., U.S. Patent Nos.
  • inhibitors of A ⁇ production see e.g., U.S. Patent Nos. 7,01 1,901 ; 6,495,540; 6,610,734; 6,632,812; 6,713,476; and 6,737,420, each incorporated herein by reference in their entireties
  • modulators of A ⁇ see e.g., U.S. Patent Nos: 6,303,567; 6,689,752, each incorporated by reference in their entireties
  • inhibitors of ⁇ -secretase see e.g., U.S. Patent Nos. 6,982,264; 7,034,182; 7,030,239, each incorporated by reference in their entireties.
  • Phage display screenings were performed to identify the proteins that interact with A ⁇ , using A ⁇ 1-42, A ⁇ 25-35, A ⁇ 12-28, and A ⁇ 1-20. To account for the possibility that the binding site of the peptides might be hindered by binding of the peptide to the polystyrene plate, a solution screening with all four peptides was also performed. BLAST searching and co-immunoprecipitation studies were then performed to identify the proteins to which the A ⁇ peptides bound.
  • Phage display screening was performed using a Ph.D. 7 peptide display system (New England Biolabs). A ⁇ 1-42, A ⁇ 1-20, A ⁇ 25-35, or A ⁇ 12-28 (37.5/cg) were coated onto an Immulon 4 HBX flat-bottom microtiter plate (Thermo Labsystems, Waltham, MA) and panned with 4 ⁇ l phage display peptide library according to the manufacturer's instructions. Bound proteins were eluted with 40 ⁇ l 0.2 M glycine pH 2.2, followed by 20 ⁇ l Tris-HCl, pH 9 and 20 ⁇ l Tris base. Four rounds of panning were performed on the plate.
  • the supernatant was aliquoted into four 17 x 100 mm Falcon 2059 tubes and incubated for 1 h at room temperature in the presence of 10 ⁇ l CuC12 or 1 ⁇ M EDTA, with 100 pg fresh biotin-LC- ⁇ -amyloid 1-42 or biotin-LC- ⁇ -amyloid 1-42 that had been preincubated at 37° for 4 days. After incubation, 0.5 ml of avidin-Agarose was added and incubation continued with shaking for 30 min.
  • the sample was cooled to 4°, transferred to a disposable column, and rapidly washed with 4 ml ice-cold homogenization buffer.
  • the proteins with affinity for A ⁇ were then sequentially eluted with 2 ⁇ l of 0.5M NaCl, 1.5M NaCl, 4M NaCl, IM Tris-HCI pH 9, or 0.2M glycine HCl pH 2.2. All buffers contained 0.1% CHAPS + 1 mM PMSF. All NaCl solutions also contained 10 mM Tris-HCI pH 7.4. The eluates were concentrated and desalted in Centricon-3 ultrafiltration units. The proteins were then separated by SDS-polyacrylamide gel electrophoresis followed by Coomassie blue staining.
  • Coomassie-stained protein hands were destained by washing with acetonitrile/acetic acid/water (1 : 1 :1), reduced, alkylated, and digested with 120 ng trypsin using the in- gel method (Rosenfeld, et al., Anal Biochem., 1992; 203, 173-179 and Hellman, et al., Anal Biochem., 1995; 224, 451-455). Digestion with trypsin was carried out overnight at 37° C. Peptides were twice extracted from the gel into 50% acetonitrile/5% formic acid.
  • the extracts were pooled, the volume reduced by vacuum centrifugation, and the final volume was brought up to 6 microliters with 25 mM ammonium bicarbonate.
  • the LC effluent was electrosprayed directly into the sampling orifice of an LTQ mass spectrometer (Thermo Finnigan) using a nanospray interface.
  • the LTQ was operated to collect MS/MS spectra in a data dependent manner, with up to five of the most intense ions that exceeded a pre-set threshold being subjected to fragmentation and analysis.
  • the MS/MS data generated were analyzed and matches to protein sequences in the NCBI non-redundant nr database (human/trypsin subset) were determined using SEQUEST (Eng, et al., J. Amer. Soc. Mass Spec. 1994; 5, 976-989) program.
  • Sequence identification was based on the cross-correlation normalized for peptide length (XConl) and delta correlation ⁇ Cn) scores. SEQUEST-derived peptide identifications and protein identifications were evaluated for statistical significance and filtered with the Peptide Prophet and Protein Prophet software tools (Nesvizhskii, et al., Anal. Chem., 2003; 75, 4646-4658). In each case, the predicted Mr and pi of the identification matched the Mr and pi values on the gel within ⁇ 5%.
  • Phage display A total of 160 clones were sequenced, yielding 61 different peptides.
  • the peptides are shown in Table 1. Although an unambiguous identification cannot be made from a seven-amino acid sequence, in most cases a mammalian-constrained BLAST search identified only a single protein, or in some cases two candidate proteins, as having a close match (Table 1).
  • the peptides found by phage display contained sequences found in phosphodiesterase 3A and 3B, and a number of proteins involved in cholesterol transport and metabolism (sortilin and steroid 5 ⁇ - reductase type 2). The strongest binding (17 clones) was to a peptide found in the P2X2 purinergic receptor and the protein Suppressor of Ty 3 homolog (SUPT3H). (Yu, et al , Genomics, 1998; 53, 90-96).
  • the co-precipitation signal with C-reactive protein precursor was very weak, because of the low levels of expression of C-reactive protein precursor in brain.
  • a ⁇ co-precipitated with the P2X2 purinergic receptor but not with CD34 or SUPT3H, indicating that peptide YQDSAKT (SEQ ID NO: 7) represented P2X2.
  • co-precipitation experiments indicated that the peptide SVLDRQR (SEQ ID NO: 8) corresponded to sortilin, and not KIDINS-220, because A ⁇ co- precipitated only with sortilin (Fig. 1); however, the sortilin band was relatively weak, suggesting low levels of sortilin in the rabbit brain.
  • LC- ⁇ -amyloid 1-42 was incubated with CHAPS extract from rabbit or mouse brain, collected with avidin-agarose, washed, and eluted with increasing salt and buffer concentrations. In one experiment, fresh and oligomerized biotin-LC- ⁇ -amyloid were also compared. Since Cu2+ has been shown to bind to 4 (Atwood, et al., J Neurochem, 2000; 75, 1219-1233 and Atwood, et al., J Biol. Chem., 1998; 273, 12817-12826), 10 ⁇ M CuC12 was also added to some samples. Rabbit brain was used because the sequence of rabbit A ⁇ is identical to that of humans. The proteins were analyzed by SDS polyacrylamide gel electrophoresis, eluted from the gel and identified by liquid chromatography / ion trap tandem mass spectrometry. The proteins are shown in Table 3.
  • peptides were dissolved in phosphate-buffered saline (PBS) at a concentration of I mM, except for peptide #3 (VSVGML WC-SEQ ID NO: 4), which was dissolved at 0.1 mM. Peptides were then sterilized by filtration before use.
  • PBS phosphate-buffered saline
  • peptide #3 VSVGML WC-SEQ ID NO: 4
  • Peptides were then sterilized by filtration before use.
  • one vial of Pro-Ject protein transduction reagent (Pierce) was then dissolved in 250 ⁇ l CHCl 3 .
  • Four ⁇ l of Pro-Ject solution were transferred to 1.5-ml polypropylene centrifuge tubes and evaporated with nitrogen.
  • Peptide was diluted in PBS to 9.1 ⁇ g/ml and 0.22 ⁇ l of peptide was added. The vial was incubated for 5 min, then vortexed, and the volume was brought
  • ⁇ -amyloid oligomerization Human A ⁇ (Anaspec 20276, 1 mg) was dissolved in 3 ml deionized water by the addition of a minimum volume of 1% ammonium hydroxide and was incubated for 3 days at 37°. The solution was then reduced in volume to 0.886 ml by lyophilization and incubated for an additional 48h at 37°. The oligomerized A ⁇ was then stored at -80°.
  • Manassas, VA were plated onto poly-L-lysine coated plates and grown at 35° in DMEM/10%FCS for several days until coverage was obtained. The cells were then induced to differentiate into a neuronal phenotype by replacing the medium with 5 ml N2 medium containing 10 ng/ml basic fibroblast growth factor at 39° C and grown in T-75 flasks at 37° C.
  • differentiated cells were grown in 12- well plates containing 1 ml N2 culture medium. When the cells reached 75-80% confluence, they were washed with serum-free DMEM and peptide/Pro-Ject mixture dissolved in 1/2 ml serum-free DMEM was added. After 4h incubation at 37°C, 1/2 ml of DMEM containing 20% fetal bovine serum was added. After 18h, oligomerized A ⁇ was added. Cells were monitored daily. After 7d, the medium was removed and the cells were washed twice with 1 x PBS. A 100 ⁇ l aliquot of PBS was added and the cells were removed by gentle scraping. The cells were then homogenized by sonication and stored at -80°.
  • ⁇ -Galactosidase Cell senescence was tested using the hydrolysis by ⁇ - galactosidase at pH 6 of 5-bromo-4-chloro-3-indolyl ⁇ -D-galactopyranoside (X-gal), a commonly-used ⁇ -galactosidase substrate. Under these conditions, ⁇ -galactosidase is easily detectable in senescent cells, but undetectable in quiescent, immortal, or tumor cells (Dimri, et al., Proc Natl Acad Sci USA, 1995; 92, 9363-9367).
  • cell homogenate (20 ⁇ l) was incubated in 100 id of 0.5M Tris-HCl, pH 6.8 containing 0.1 mg/ml X-gal (5-bromo-4-chloro-3-indolyl-( ⁇ -D-galactopyranoside). After 24h at 37°C, the samples were diluted to 1 ml and absorbance at 610 nm was measured.
  • pBad is a member of the Bcl-2 family, and regulates the survival signal (of Bc 12, P. and regulation of apoptosis, Leukemia, 2001 ; 15, 515-522).
  • Unphosphorylated Bad dimerizes with Bcl-2 and BcI-XL, which neutralizes their anti- apoptotic activity.
  • Activation of the phosphoinositol 3-kinase pathway ultimately leads to activation of Akt, which phosphorylates Bad on serine 136.
  • Activation of MAP kinase pathways results in phosphorylation of Bad on serine 1 12.
  • Phosphorylated Bad is sequestered from its proapoptotic role by binding with 14-3-3 protein (Masters, et al., Biochem Soc Trans., 2002; 30, 360-36516). Thus, a decrease in Bad phosphorylation indicates apoptosis.
  • pBad was measured by densitometry of Western blots stained with phospho-Bad antibody. Image quantitation and molecular weight estimation were done on 16-bit images using the Unix-based image analysis program imal (http://brneurosci.org/imal.html).
  • the background value for each band was calculated by fuzzy k-means clustering analysis of the appropriate region of the image (Kaufman, et al., Finding groups in data: an introduction to cluster analysis, Wiley-Interscience, New York, (1990); Bezdek, J. C, Pattern recognition with fuzzy objective function algorithms, Plenum, New York, (1981); Bezdek, J. C, J. Cybern., 1971 ; 3, 58; and Dunn, J. C, J. Cybern., 1974; 3, 32). Densitometry results are expressed as units of relative staining.
  • Caspase 3 assay Members of the caspase family of cysteine aspartyl proteases are related to the C. elegans CED-3 death protein. Caspases 8, 9, and 10 are activated by receptor clustering and are known as “initiator caspases” (Riedl, et al., Nat Rev MoI Cell Biol, 2004; 5, 897-907). Caspases 3, 6, and 8 are activated by changes in mitochondrial permeability that are associated with apoptosis, and are known as “effector caspases”. Effector caspase 3 proteolyzes a number of substrates, including DFF45/ICAD, PARP, gelsolin, and nuclear lamins 1221.
  • Proteolysis of DFF45/ICAD liberates the DNAse subunit of DFF to cause chromatin degradation (Wolf, et al., J Biol Chem., 1999; 274, 30651-30656).
  • Proteolysis of PARP has been used by many researchers as a marker for apoptosis. Caspase 3 activity was measured fluorometrically using the commercially-available substrate Ac-Asp-Glu-Val-Asp-NH2-methylcoumarin, which contains the PARP cleavage site (Fernandes-Alnemri, et al., Cancer Res., 1995; 55, 6045-6052). Hydrolysis of this peptide yields a species that fluoresces at 440-460 nm.
  • Phosphodiesterase was measured by the method of Alvarez and Daniels (Alvarez, et al., Anal Biochem., 1992; 203, 76-82) with slight modifications. Fines were removed from acidic alumina (Brockmann grade 1) by resuspending several times in water. Bio-Rad Poly-Prep columns were packed with a slurry containing 1.3 g alumina to a height of 3 cm. Columns were precycled with 0.1 M ammonium acetate to remove any cAMP, then equilibrated with water.
  • rat brain homogenate was diluted to 100 ⁇ l with water and 10 ⁇ l buffer (I M Tris-HCl pH 7.5 2 mM MgSO4) and 0.5 ⁇ l 3H-cAMP were added. After 10 min incubation at 30° C, the samples were boiled and cooled and 0.1 Al 5'-nucleotidase (Biomol, 500 kU/ml). The samples were incubated for 30 min at 30° C and applied to the column. The adenosine was eluted with 4 ml 5 mM HCl, mixed with scintillation fluid, and counted in a scintillation counter.
  • the cells were collected after 7 days in culture and analyzed for markers of apoptosis (caspase 3 and pBad) and senescence ( ⁇ - galactosidase).
  • the peptide sequences are shown in Table 4.
  • Table 4 Sequences of A ⁇ -binding peptides tested in cultured cells for their ability to protect against A ⁇ . The binding region on A13 found by phage display is also shown. The probable identity of each peptide was determined by BLAST, using co-precipitation studies to discriminate between high-scoring proteins.
  • Phosphorylated Bad was significantly decreased in H19-7/IGF-IR cells after A ⁇ 1 -42 treatment (Fig. 4A), indicating early stages of apoptosis.
  • Peptides #3 (VSVGMLWC; SEQ ID NO. 4), representing steroid 5- ⁇ reductase) and #4 (SVLDRQRC; SEQ ID NO: 5), representing sortilin-related receptor) prevented the decrease, with #3 almost completely blocking the decrease.
  • peptides #3, #4, and #5 (LGSYKPSC; SEQ ID NO: 6), representing ERGIC2) also prevented the increase in caspase activity produced by A ⁇ 1-42 treatment (Fig. 4B), with peptides #3 and #5 being equally potent at blocking caspase activation.
  • Peptides #4 and #5 also reduced the increase in ⁇ -galactosidase produced by A ⁇ 1 -42 treatment (Fig. 4C), indicating that they were partially protective against A ⁇ -induced senescence.
  • peptides #1 YQDSAKTC; SEQ ID NO: 10, P2X2 purinergic receptor
  • #2 NDRGLLAC; SEQ ID NO: 1 1, TMS Membrane protein
  • the three peptides with the strongest beneficial effect corresponded to sortilin, steroid-5 ⁇ reductase, and ERGIC2, A ⁇ -interacting proteins that are involved in cholesterol, LDL, or ER membrane transport.
  • the binding region for phosphodiesterase and steroid reductase can be narrowed down to amino acids 12-20 of A ⁇ (VHHQKLVFF; SEQ ID NO: 28).
  • the binding sites for sortilin and ApoB are within the region of amino acids 25-25
  • the binding region for P2X2 is within the region of amino acids 12-28 (Fig. 5).
  • This region includes a PKC pseudo-phosphorylation site (amino acids 26- 30, SNKGA; SEQ ID NO: 3).
  • a ⁇ 1-42 competitively inhibits cholesterol binding to ApoE and low-density lipoprotein (Yao, et al., FASEB J., 2002; 16, 1677-1679).
  • ERGIC-53, ERGIC2, sortilin, P2X2, ApoB, steroid reductase, and the C-reactive protein precursor The interactions with 14-3-3 and sortilin are of particular relevance to Alzheimer's Disease. Sortilin expression has been shown to correlate inversely with AD neuropathology. Inherited variants in the sortilin SORL l (LR II) lipoprotein receptor, which mediates a variety of cellular sorting and trafficking functions, are genetically associated with late-onset AD (Rogaeva, et al. , Nat Genet., 2000; 39,68-77).

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Abstract

La présente invention concerne des peptides qui se lient à des protéines et réduisent l'apoptose médiée par Aβ (39- 42) dans les cellules neuronales, par rapport aux cellules neuronales non traitées. La présente invention concerne également une composition pharmaceutique comprenant ces peptides et des anticorps contre les peptides précédents.
PCT/US2008/008147 2007-06-28 2008-06-30 Peptides, compositions et procédés pour réduire l'apoptose médiée par les bêta-amyloïdes WO2009005783A1 (fr)

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103619356A (zh) * 2011-05-05 2014-03-05 萨勒普塔医疗公司 肽寡核苷酸缀合物
CN103619356B (zh) * 2011-05-05 2017-09-12 萨勒普塔医疗公司 肽寡核苷酸缀合物

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