WO2011011181A1 - Chaperons pharmacologiques pour le traitement de la maladie d’alzheimer - Google Patents

Chaperons pharmacologiques pour le traitement de la maladie d’alzheimer Download PDF

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WO2011011181A1
WO2011011181A1 PCT/US2010/040774 US2010040774W WO2011011181A1 WO 2011011181 A1 WO2011011181 A1 WO 2011011181A1 US 2010040774 W US2010040774 W US 2010040774W WO 2011011181 A1 WO2011011181 A1 WO 2011011181A1
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secretase
protein
activity
cell
batimastat
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Robert Boyd
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Amicus Therapeutics, Inc.
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    • 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
    • A61K38/55—Protease inhibitors
    • A61K38/57—Protease inhibitors from animals; from humans
    • 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
    • A61K31/38—Heterocyclic compounds having sulfur as a ring hetero atom
    • A61K31/381—Heterocyclic compounds having sulfur as a ring hetero atom having five-membered rings

Definitions

  • the present invention relates to a method for treating an individual having a neurological disorder with an associated increase in the ⁇ -amyloidogenic metabolism of amyloid precursor protein (APP).
  • the individual is administered a pharmacological chaperone for ⁇ -secretase, which increases trafficking of the ⁇ -secretase protein from the ER to the cytoplasm in neural cells, concomitantly increasing ⁇ -secretase enzyme activity in neural cells.
  • APP amyloid precursor protein
  • AD Alzheimer's disease
  • a chronic and progressive neurodegenerative disorder characterized neuropathologically by the presence of amyloid- ⁇ plaques, neurofibrillary tangles, and gray matter loss.
  • multiple regions of brain gray matter have a profound neuronal loss, including basal forebrain, hippocampus, entorhinal, and temporal cortices.
  • Neurofibrillary tangles are composed of an abnormally hyperphosphorylated intracellular protein called Tau, tightly wound into paired helical filaments and thought to impact microtubule assembly and protein trafficking, resulting in the eventual demise of neuronal viability.
  • the extracellular amyloid- ⁇ plaque deposits are composed of a proteinacious core of insoluble aggregated amyloid- ⁇ (A ⁇ l -42) peptide and have 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.
  • AD Alzheimer's disease
  • AD is set to become the developed world's largest socioeconomic healthcare burden over the coming decades. AD is thought to affect 4-8% of the population over 65 years of age, with the incidence continuing to increase with increasing age.
  • Antemortem clinical diagnosis of AD is difficult and requires a recorded decline in cognitive function as well as evidence of progressive deficits in other behavioral areas such as executive function and language skills. Unqualified diagnosis of AD can still only be made neuropathologically postmortem by examination of patients' brains and the detection of amyloid- ⁇ plaques and tangles.
  • AD cases The majority of Alzheimer cases seem to be sporadic or to result from complex interactions of several genes. Nevertheless, a minority of AD cases ( ⁇ 1%) result from autosomal dominant inheritance of an age-dependent trait with high penetrance (Tanzi, R. E., et al., Neuron 2001 ; 32: 181-184; St George-Hyslop, P.H., Ann NYAcad Sci 2000; 924: 1-7).
  • a ⁇ is a hydrophobic 39- to 42-amino acid peptide, found in all biological fluids, and derived from the enzymatic cleavage of a larger type I membrane protein, the amyloid precursor protein (APP) (Hardy, J., et al., Science 200; 297: 353-356).
  • APP amyloid precursor protein
  • APOE4 apolipoprotein E4
  • the ⁇ -amiloydogenic pathway involves the sequential proteolysis of APP by ⁇ - secretase (BACE) followed by ⁇ -secretase. Although this is a minor APP processing route, it is this pathway that generates A ⁇ fragments believed to give rise to AD.
  • BACE-I and BACE-2 two ⁇ - secretase genes have been identified, referred to as BACE-I and BACE-2, colocalized with APP in the endosomal compartment (Vassar, R. et al., Science 1999; 286: 735-741 ). Whereas both can process APP at the same site, only BACE-I is significantly expressed in brain, particularly in neurons, indicating that neurons are the major source of amyloid- ⁇ peptides in brain.
  • BACE-2 is expressed in heart, kidney, and placenta
  • drugs developed as ⁇ -secretase inhibitors may need to be selective against BACE-2 to prevent unwanted peripheral side effects in the clinic
  • the predominant pathway by which APP is processed does not give rise to A ⁇ fragments, and hence is referred to as the non- ⁇ -amyloidogenic pathway.
  • the initial APP processing involves the cleavage of APP by ⁇ -secretase.
  • the identification of proteins with ⁇ -secretase activity is ongoing, and currently includes three members of the A Disintegrin And Metal loproteinase (ADAM) protein family.
  • ADAM A Disintegrin And Metal loproteinase
  • ADAMl O also known as TACE (tumor necrosis factor- ⁇ converting enzyme)
  • ADAM9 Yamamoto S., et al., Proc Natl Acad Sci USA 1999; 96: 3922-3927; Zheng Y., et al., J Biol Chem 2004; 279: 42898 ⁇ 2906; Kowalska, A., Pol. J. Pharmacol 2004; 56: 171-178).
  • ⁇ -secretase cleavage site is within the A ⁇ sequence of APP, and none of the proteolytic fragments created by ⁇ -secretase cleavage have been associated with the generation of AD, enhanced cleavage at this site could represent a disease modifying strategy for AD.
  • AD amyloid- ⁇ fragment
  • a ⁇ amyloid- ⁇ fragment
  • AD Alzheimer's Disease
  • AD therapies As such, blockade of the NMDA receptor, one of the principal excitatory glutamate receptors in the brain, has been shown to have neuroprotective effects in a number of acute preclinical in vitro and in vivo models. Additional research to develop AD therapies has focused on inhibition of ⁇ -secretase and the metabolism of APP to form A ⁇ peptide.
  • An alternative approach to developing treatments for AD may focus on the shunting of APP into the non- ⁇ -amyloidogenic metabolism of APP into sAPP ⁇ by ⁇ -secretase.
  • the use of pharmacological inhibitors to function as pharmacological chaperones, increasing the enzymatic activity of misfolded mutant proteins, as well as normal wild type proteins has been demonstrated (see U.S. Patent Nos. 6,274,597; 6,583,158; 6,589,964; 6,599,919; and 6,916,829, all incorporated herein by reference).
  • proteins are involved in almost every aspect of cellular function. Proteins are linear strings of amino acids that fold and twist into specific three-dimensional shapes in order to function properly. Certain human diseases result from mutations that cause changes in the amino acid sequence of a protein which reduce its stability and may prevent it from folding properly. The majority of genetic mutations that lead to the production of less stable or misfolded proteins are called missense mutations. These mutations result in the substitution of a single amino acid for another in the protein. Because of this error, missense mutations often result in proteins that have a reduced level of biological activity. In addition to missense mutations, there are also other types of mutations that can result in proteins with reduced biological activity.
  • Proteins generally fold in a specific region of the cell known as the endoplasmic reticulum, or ER.
  • the cell has quality control mechanisms that ensure that proteins are folded into their correct three-dimensional shape before they can move from the ER to the appropriate destination in the cell, a process generally referred to as protein trafficking. Misfolded proteins are often eliminated by the quality control mechanisms after initially being retained in the ER. In certain instances, misfolded proteins can accumulate in the ER before being eliminated.
  • misfolded proteins in the ER interrupts their proper trafficking, and the resulting reduced biological activity can lead to impaired cellular function and ultimately to disease.
  • the accumulation of misfolded proteins in the ER may lead to various types of stress on cells, which may also contribute to cellular dysfunction and disease.
  • Endogenous molecular chaperones are present in virtually all types of cells and in most cellular compartments. Some are involved in the transport of proteins and permit cells to survive under stresses such as heat shock and glucose starvation (Gething et al., Nature 1992; 355:33-45; Caplan, Trends Cell. Biol. 1999; 9:262-268; Lin et al, MoI. Biol. Cell 1993; 4.109-1119; Bergeron et al., Trends Biochem. Sci. 1994; 19:124-128).
  • BiP immunoglobulin heavy-chain binding protein, Grp78
  • Grp78 is the best characterized chaperone of the ER (Haas, Curr. Top. Microbiol.
  • BiP Like other chaperones, BiP interacts with many secretory and membrane proteins within the ER throughout their maturation. When nascent protein folding proceeds smoothly, this interaction is normally weak and short-lived. Once the native protein conformation is achieved, the molecular chaperone no longer interacts with the protein. BiP binding to a protein that fails to fold, assemble, or be properly glycosylated becomes stable, and usually leads to degradation of the protein through the ER-associated degradation pathway.
  • the pharmacological chaperones enhance ER secretion and activity of wild-type enzymes (see Examples).
  • a compound that induces a stable molecular conformation of an enzyme during folding serves as a "chaperone" to stabilize the enzyme in a proper conformation for exit from the ER.
  • a compound that induces a stable molecular conformation of an enzyme during folding serves as a "chaperone" to stabilize the enzyme in a proper conformation for exit from the ER.
  • enzymes one such compound unexpectedly turned out to be a competitive inhibitor of the enzyme.
  • Stabilization of these proteins also may be achieved using pharmacological chaperones.
  • small synthetic compounds were found to stabilize the D ⁇ A binding domain of mutant forms of the tumor suppressor protein p53, thereby allowing the protein to maintain an active conformation (Foster et al., Science 1999; 286:2507-10).
  • Synthesis of receptors has been shown to be rescued by small molecule receptor antagonists and ligands (Morello et al., J. Clin. Invest. 2000; 105: 887-95; Petaja- Repo et al., EMBO J. 2002; 21 :1628-37).
  • the present invention provides a method for the treatment of a neurological disorder in an individual, wherein the neurological disorder is associated with the ⁇ -amyloidogenic processing of Amyloid Precursor Protein (APP), by administering an effective amount of a specific pharmacological chaperone to treat the neurological disorder.
  • the individual has been diagnosed or is at risk of developing Alzheimer's disease (AD), including Familial or Sporadic forms of AD.
  • AD Alzheimer's disease
  • the present invention provides a method for enhancing intracellular folding of an ⁇ -secretase polypeptide into a functional conformation by contacting an ⁇ -secretase -expressing cell with an effective amount of a specific pharmacological chaperone. Enhancing intracellular folding of ⁇ -secretase will lead to an increased proportion of a-secretase which exits the ER, resulting in enhanced enzymatic function in the cells of the central nervous system. Increased ⁇ -secretase activity would, in turn, increase the non- ⁇ -amyloidogenic metabolism of APP, and may be useful in the treatment of neurological disorders such as AD.
  • the ⁇ -secretase polypeptide is a wild-type ⁇ -secretase polypeptide, which, for example, is encoded by a nucleic acid of the zinc metalloproteinase adamalysin family of A Disintegrin And Metalloproteases (ADAM).
  • ADAMs include, but are not limited to, ADAM 9, ADAM 10, and TACE/ADAM 17.
  • the ⁇ -secretase polypeptide is a mutant ⁇ -secretase polypeptide
  • the mutant polypeptide contains a mutation that results in reduced or improper intracellular folding of the ⁇ -secretase polypeptide, resulting in a shift of equilibrium to increased ⁇ -amyloidogenic processing of APP
  • a pharmacological chaperone specific for ⁇ -secretase can rescue the mutant ⁇ -secretase.
  • the pharmacological chaperone binds the ⁇ -secretase active site. In another embodiment, the pharmacological chaperone binds a non-active site of ⁇ -secretase, which can include an allosteric site.
  • the method of the invention comprises the administration of one or more pharmacological chaperones of ⁇ -secretase to an individual diagnosed, at risk, or suspected to have Alzheimer's Disease. Suitable pharmacological chaperones include any compound(s) which, following administration to an individual, will bind to ⁇ -secretase and increase ⁇ -secretase enzymatic activity. In one particular embodiments, the pharmacological chaperone is a reversible inhibitor of ⁇ -secretase.
  • the increase in ⁇ -secretase enzymatic activity may increase the non- ⁇ -amyloidogenic processing of Amyloid Precursor Protein, producing sAPP ⁇ , and decrease the ⁇ -amyloidogenic processing of Amyloid Precursor Protein, reducing the accumulation of amyloid- ⁇ fragment, particularly the A ⁇ l-42 peptide, within the central nervous system of an individual.
  • pharmacological inhibitors of ⁇ -secretase as ⁇ -secretase chaperones can increase the wild-type enzymatic activity of ⁇ -secretase.
  • Pharmacological inhibitors of ⁇ secretase include, but are not limited to, hydroxamic acid-based zinc metalloproteinase inhibitors, for example, batimastat. SB223820, marimastat, BB3103, BB3132, TAPI-O, TAPI-I , TAPI-2, Immunex compound 3 (IC3), KD-IX-73-4, BB21 16, and analogs thereof.
  • Figure 1 shows mean ⁇ -galactosidase A activity in white blood cells from normal, healthy volunteers who received 50 mg 1-deoxygalactonojirimycin (DGJ) b.i.d. (triangles), 150 mg DGJ b.i.d. (squares), or placebo (open circles).
  • Figure 2 shows mean ⁇ -galactosidase A activity in white blood cells from 1 1 Fabry disease patients who were treated with DGJ.
  • DGJ 1-deoxygalactonojirimycin
  • Figure 3 shows the pharmacological chaperones TAPI-2, GM6001 and GW4023 stabilize purified ADAMlO in vitro and increases wild-type levels of ADAMlO (precursor and mature forms) by 2- to 3-fold in SY5Y neuroblastomas.
  • the invention is based, in part, on the discovery that administration of a pharmacological chaperone to a human resulted in a meaningful increase in the level of activity of a wild-type protein.
  • This discovery combined with an understanding of a pharmacological chaperone's ability to promote proper protein folding in the ER, leading to correct protein trafficking and, significantly increased protein activity, e.g., sufficient protein activity to reverse or ameliorate a disease, disorder, or condition in a human subject.
  • This phenomenon is highly specific to the protein specifically bound by the particular pharmacological chaperone, in contrast to methods using compounds that operate generally to increase expression of all proteins, called "chemical chaperones.”
  • pharmacological chaperones increased endogenous wild-type protein activity in humans to about 120% of normal, 130% of normal, and 145% of normal at a lower dose, and to 150% and 185% of normal at a higher dose after administration of a pharmacological chaperone (see Example 1 and Figure 1).
  • This level of increase in vivo was not predictable from results with cells in tissue culture which remain exposed to the pharmacological chaperone.
  • U.S. Patent No. 6,274,597 describes a 30% increase of ⁇ -galactosidase A ( ⁇ -GAL) activity in normal lymphoblasts cultured with deoxygalactonojirimycin (DGJ), a pharmacological chaperone.
  • ⁇ -GAL ⁇ -galactosidase A
  • Example 10 of U.S. Patent No. 6,274,597 describes an increase in activity of a mutant enzyme in transgenic mice treated for one week with a pharmacological chaperone.
  • these experiments involved mutant forms of the rescued protein, not wild-type, and were conducted in mice, so the results were not predictive or suggestive of the results observed for wild-type protein in humans.
  • a pharmacological chaperone could increase the level of activity of a wild-type protein in vivo by at least 20-25%, and particularly not by at least about 50%.
  • administration of DGJ to subjects resulted in a dose-dependent increase in ⁇ -GAL activity.
  • the level of increase in enzyme activity increased by at least 50% (at least 1.5-fold) to up to 100% (at least 2-fold).
  • This extraordinary effect results from titrating a pharmacological chaperone, which is already demonstrated in accordance with existing technology, to rescue a mutant form of the protein, in human patients, to achieve this level of increase, as described herein.
  • the invention provides for titrating a dose of a pharmacological chaperone that has been found to rescue activity of a mutant protein to increase the level of activity of a wild-type protein.
  • the term "pharmacological chaperone,” or sometimes “specific pharmacological chaperone” (“SPC”) refers to a molecule that specifically binds to ⁇ - secretase and has one or more of the following effects: (i) enhancing the formation of a stable molecular conformation of the protein; (ii) enhances proper trafficking of the protein from the ER to another cellular location, preferably a native cellular location, i.e., preventing ER- associated degradation of the protein; (iii) preventing aggregation of conformational Iy unstable, i.e., misfolded proteins; (iv) restoring or enhancing at least partial wild-type function, stability, and/or activity of the protein; and/or (v) improving the phenotype or function of the cell harboring ⁇ -secretase.
  • SPC specific pharmacological chaperone
  • a pharmacological chaperone for ⁇ -secretase is a molecule that binds to ⁇ -secretase, resulting in proper folding, trafficking, non- aggregation, and activity of ⁇ -secretase.
  • this term does not refer to endogenous chaperones, such as BiP, or to non-specific agents which have demonstrated nonspecific chaperone activity against various proteins, such as glycerol, DMSO or deuterated water, i.e., chemical chaperones (see Welch et al , Cell Stress and Chaperones 1996; l(2): 109-1 15; Welch et al., Journal of Bioenergetics and Biomembranes 1997; 29(5):491- 502; U.S. Patent No. 5,900,360; U.S. Patent No. 6,270,954; and U.S. Patent No. 6,541 ,195).
  • specific binding molecules e.g., active site-specific chaperones (discussed above), inhibitors or antagonists, and agonists.
  • specifically binds refers to the interaction of a pharmacological chaperone with ⁇ -secretase, specifically, an interaction with amino acid residues of ⁇ -secretase that directly participate in contacting the pharmacological chaperone.
  • a pharmacological chaperone specifically binds a target protein, e.g., ⁇ -secretase, to exert a chaperone effect on ⁇ -secretase and not a generic group of related or unrelated proteins.
  • amino acid residues of ⁇ -secretase that interact with any given pharmacological chaperone may or may not be within the protein's "active site.” Specific binding can be evaluated through routine binding assays or through structural studies, e.g., co-crystallization, NMR, and the like.
  • the pharmacological chaperone is an inhibitor or antagonist of ⁇ -secretase. In another non-limiting embodiment, the pharmacological chaperone is an agonist of ⁇ -secretase. In yet another embodiment, the pharmacological chaperone is a mixed agonist/antagonist.
  • the term "antagonist” refers to any molecule that binds to a protein and either partially or completely blocks, inhibits, reduces, or neutralizes an activity of ⁇ -secretase.
  • agonist refers to any molecule that binds to a protein and at least partially increases, enhances, restores, or mimics an activity of ⁇ - secretase. As discussed below, such molecules are known for ⁇ -secretase.
  • the terms "enhance ⁇ -secretase conformational stability" or “increase ⁇ -secretase conformational stability” refer to increasing the amount or proportion of ⁇ - secretase that adopts a functional conformation in a cell contacted with a pharmacological chaperone specific for ⁇ -secretase, relative to ⁇ -secretase in a cell (preferably of the same cell-type or the same cell, e.g., at an earlier time) not contacted with the pharmacological chaperone specific for ⁇ -secretase.
  • the cells do not express a conformation mutant ⁇ -secretase.
  • the cells do express a mutant ⁇ - secretase polynucleotide encoding a polypeptide e.g., a conformational mutant ⁇ -secretase.
  • the terms "enhance ⁇ -secretase trafficking" or “increase ⁇ -secretase trafficking” refer to increasing the efficiency of transport of ⁇ -secretase into the cytosol of a cell contacted with a pharmacological chaperone specific for ⁇ -secretase, relative to the efficiency of transport of ⁇ -secretase in a cell (preferably of the same cell-type or the same cell, e.g., at an earlier time) not contacted with the pharmacological chaperone specific for ⁇ - secretase.
  • the terms "enhance ⁇ -secretase activity" or “increase ⁇ -secretase activity” refer to increasing the activity of ⁇ -secretase, as described herein, in a cell contacted with a pharmacological chaperone specific for ⁇ -secretase, relative to the activity of ⁇ - secretase in a cell (preferably of the same cell-type or the same cell, e.g., at an earlier time) not contacted with the pharmacological chaperone specific for ⁇ -secretase.
  • the terms "enhance ⁇ -secretase level" or “increase ⁇ -secretase level” refer to increasing the level of ⁇ -secretase in a cell contacted with a pharmacological chaperone specific for ⁇ -secretase, relative to the level of ⁇ -secretase in a cell (preferably of the same cell-type or the same cell, e.g., at an earlier time) not contacted with the pharmacological chaperone specific for ⁇ -secretase.
  • stabilize a proper conformation refers to the ability of an ⁇ -secretase pharmacological chaperone to induce or stabilize a conformation of a mutated ⁇ -secretase protein that is functionally identical to the conformation of the wild-type ⁇ -secretase protein.
  • conformational flexibility does not result in (1) protein aggregation, (2) elimination through the endoplasmic reticulum-associated degradation pathway, (3) impairment of protein function, e.g., APP metabolic activity, and/or (4) improper transport within the cell, e.g., localization to the cytosol, to a greater or lesser degree than that of the wild-type protein.
  • stable molecular conformation refers to a conformation of a protein, i.e., ⁇ -secretase, induced by a pharmacological chaperone that provides at least partial wild-type function in the cell.
  • a stable molecular conformation of a mutant ⁇ -secretase would be one where ⁇ -secretase escapes from the ER and traffics to the cytosol as for a wild- type ⁇ -secretase, instead of misfolding and being degraded.
  • a stable molecular conformation of a mutated ⁇ -secretase may also possess full or partial ⁇ -secretase activity, e.g., APP metabolism.
  • it is not necessary that the stable molecular conformation have all of the functional attributes of the wild-type protein.
  • ⁇ -secretase activity refers to the normal physiological function of a wild- type ⁇ -secretase in a cell.
  • ⁇ -secretase activity includes metabolism of APP.
  • Such functionality can be tested by any means known to establish functionality.
  • wild-type ⁇ -secretase or " ⁇ -secretase” refer to a polypeptide encoded by a nucleic acid selected from the zinc metalloproteinase adamalysin family of A Disintegrin And Metalloproteases (ADAM) proteins comprising ADAM 9 (human ADAM9, GenBank Accession Nos. NM 003816, and NM 001005845; murine ADAM9, GenBank Accession No. NM_007404; and predicted rat ADAM9, GenBank Accession No. NMJ)Ol 014772), ADAM 10 (human ADAMl O, GenBank Accession No. NMJ)Ol I l O; murine ADAMlO, GenBank Accession No.
  • ADAM 9 human ADAM9, GenBank Accession Nos. NM 003816, and NM 001005845
  • murine ADAM9 GenBank Accession No. NM_007404
  • predicted rat ADAM9 GenBank Accession No. NMJ
  • ⁇ -secretase is encoded by ADAMlO (Obregon et al., J. Biol. Chem. 2006; 281 : 16419-16427).
  • ⁇ -secretase polypeptide may be encoded for by any nucleic acid molecule exhibiting 50%, 60%, 70%, 80% and up to 100% homology to the nucleic acid molecules encoding human ⁇ -secretase, and any sequences which hybridize under standard conditions to these sequences.
  • any other nucleotide sequence that encodes ⁇ -secretase polypeptide such as allelic variants in normal individuals, that have the ability to achieve a functional conformation in the ER, achieve proper localization within the cell, and exhibit wild-type activity (e.g., APP metabolism).
  • ⁇ -secretase polypeptide also refers to an amino acid sequence depicted from the ADAM family of proteins comprised of: ADAM 9 (human ADAM9, GenBank Accession Nos. NP 001005845, and NP 003807; murine ADAM9, GenBank Accession No. NP 031430; and predicted rat ADAM9, GenBank Accession No. NPJ)Ol 014772), ADAMlO (human ADAMl O, GenBank Accession No. NPJ)OI lOl ; murine ADAMl O, GenBank Accession No. NP 031425, and predicted rat ADAMlO, GenBank Accession No.
  • TACE/ADAM 17 human ADAM 17, GenBank Accession No. NP_003174; murine ADAM17, GenBank Accession No. NP_033745; and rat ADAM17, GenBank Accession No. NP_064702
  • mutant ⁇ -secretase refers to an ⁇ -secretase polypeptide translated from a gene containing a genetic mutation that results in an altered ⁇ -secretase amino acid sequence.
  • the mutation results in an ⁇ -secretase protein that does not achieve a native conformation under the conditions normally present in the ER, when compared with wild-type ⁇ -secretase, or exhibits decreased stability or activity as compared with wild-type ⁇ -secretase.
  • a mutation may occur in a non-coding part of the gene encoding ⁇ -secretase that results in less efficient expression of the protein, e.g., a mutation that affects transcription efficiency, splicing efficiency, mRNA stability, and the like.
  • Certain tests may evaluate attributes of a protein that may or may not correspond to its actual in vivo activity, but nevertheless are appropriate surrogates of protein functionality, and wild-type behavior in such tests demonstrates evidence to support the protein folding rescue or enhancement techniques of the invention.
  • One such activity in accordance with the invention is appropriate transport of a functional ⁇ -secretase from the endoplasmic reticulum to the cytosol.
  • endogenous expression and “endogenously expressed” refers to the normal physiological expression of ⁇ -secretase in cells in an individual not having or suspected of having a disease or disorder associated with ⁇ -secretase deficiency, overexpression of a dominant negative mutant, or other defect, such as a mutation in ⁇ - secretase nucleic acid or polypeptide sequence that alters, e.g., inhibits, its expression, activity, or stability.
  • This term also refers to the expression of ⁇ -secretase in cells or cell types in which it is normally expressed in healthy individuals, and does not include expression of ⁇ -secretase in cells or cell types, e.g., tumor cells, in which ⁇ -secretase is not expressed in healthy individuals.
  • the term "efficiency of transport” refers to the ability of a protein to be transported out of the endoplasmic reticulum to its native location within the cell, cell membrane, or into the extracellular environment.
  • a “competitive inhibitor" of an enzyme can refer to a compound which structurally resembles the chemical structure and molecular geometry of the enzyme substrate to bind the enzyme in approximately the same location as the substrate.
  • the inhibitor competes for the same active site as the substrate molecule, thus increasing the Km.
  • Competitive inhibition is usually reversible if sufficient substrate molecules are available to displace the inhibitor, i.e., competitive inhibitors can bind reversibly. Therefore, the amount of enzyme inhibition depends upon the inhibitor concentration, substrate concentration, and the relative affinities of the inhibitor and substrate for the active site.
  • Non-classical competitive inhibition occurs when the inhibitor binds remotely to the active site, creating a conformational change in the enzyme such that the substrate can no longer bind to it.
  • the binding of substrate at the active site prevents the binding of inhibitor at a separate site and vice versa. This includes allosteric inhibition.
  • a "linear mixed-type inhibitor" of an enzyme is a type of competitive inhibitor that allows the substrate to bind, but reduces its affinity, so the Km is increased and the Vmax is decreased.
  • non-competitive inhibitor refers to a compound that forms strong bonds with an enzyme and may not be displaced by the addition of excess substrate, i.e., non-competitive inhibitors may be irreversible.
  • a non-competitive inhibitor may bind at, near, or remote from the active site of an enzyme or protein, and in connection with enzymes, has no effect on the Km but decreases the Vmax.
  • Uncompetitive inhibition refers to a situation in which inhibitor binds only to the enzyme-substrate (ES) complex. The enzyme becomes inactive when inhibitor binds. This differs from non-classical competitive inhibitors which can bind to the enzyme in the absence of substrate.
  • Vmax refers to the maximum initial velocity of an enzyme catalyzed reaction, i.e., at saturating substrate levels.
  • Km is the substrate concentration required to achieve /2 Vmax.
  • An enzyme “enhancer” is a compound that binds to ⁇ -secretase and increases the enzymatic reaction rate.
  • terapéuticaally effective dose and “effective amount” refer to an amount sufficient to enhance protein processing in the ER (permitting a functional conformation), without inhibiting protein already expressed at the appropriate cellular location (in the case of an antagonist), or without inducing ligand-mediated receptor internalization of protein from the appropriate cellular location (in the case of an agonist), and enhance activity of the target protein, thus resulting in a therapeutic response in a subject.
  • a therapeutic response may be any response that a user (e.g., a clinician) will recognize as an effective response to the therapy, including the foregoing symptoms and surrogate clinical markers.
  • a therapeutic response will generally be an amelioration or inhibition of one or more symptoms of a disease or disorder, e.g., Alzheimer's Disease.
  • 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 the compound 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.
  • 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.
  • an isolated nucleic acid 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 nucleic acid includes a PCR product, an mRNA band on a gel, a cDNA, or a restriction fragment.
  • an isolated nucleic acid is preferably excised from the chromosome in which it may be found, and more preferably is no longer joined to non-regulatory, non-coding regions, or to other genes, located upstream or downstream of the gene contained by the isolated nucleic acid molecule when found in the chromosome.
  • the isolated nucleic acid lacks one or more introns.
  • Isolated nucleic acids include sequences inserted into plasmids, cosmids, artificial chromosomes, and the like.
  • a recombinant nucleic acid is an isolated nucleic acid.
  • An isolated protein may be associated with other proteins or nucleic acids, or both, with which it associates in the cell, or with cellular membranes if it is a membrane-associated protein.
  • An isolated organelle, cell, or tissue is removed from the anatomical site in which it is found in an organism.
  • An isolated material may be, but need not be, purified.
  • purified refers to material, such as a ⁇ -secretase nucleic acid or polypeptide that has been isolated under conditions that reduce or eliminate unrelated materials, i.e., contaminants.
  • a purified protein 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.
  • AD Alzheimer's Disease
  • a ⁇ l-42 amyloid- ⁇
  • amyloid- ⁇ particularly A ⁇ l-42
  • peptide is formed from the ⁇ - amyloidogenic metabolism of APP.
  • AD may be heritable in a Familial manifestation, or may be sporadic.
  • AD includes Familial, Sporadic, as well as intermediates and subgroups thereof based on phenotypic manifestations. Familial AD typically has an early-onset (before age 65) while Sporadic AD typically is late-onset (age 65 and later).
  • Familial AD may be associated with mutations in one or more genes selected from the group comprising presenilin 1 (human presenilin 1, GenBank Accession Nos. NM 000021 , NM 007318, and NM 007319; murine presenilin 1, GenBank Accession No. NM 008943; and rat presenilin 1, GenBank Accession No. NM 019163), presenilin 2 (human presenilin 2, GenBank Accession Nos. NM 000447, and NM_012486; murine presenilin 2, GenBank Accession No. NM Ol 1 183; and rat presenilin 2, GenBank Accession No.
  • presenilin 1 human presenilin 1, GenBank Accession Nos. NM 000021 , NM 007318, and NM 007319
  • murine presenilin 1 GenBank Accession No. NM 008943 GenBank Accession No. NM 019163
  • presenilin 2 human presenilin
  • NM 031087) Amyloid Precursor Protein (APP) (human APP, GenBank Accession Nos. NM_201414, NM_201413, and NM_000484; murine APP, GenBank Accession No. NM_007471 ; and rat APP, GenBank Accession No. NM O 19288).
  • Sporadic AD can not be tested for directly, but certain risk factors may increase an individual's susceptibility to developing sporadic AD.
  • individuals with at least one copy of the e4 allele of Apolipoprotein E (APOE) (human APOE, GenBank Accession No. NM_000041 ; murine APOE, GenBank Accession No. NM_009696; and rat APOE, GenBank Accession No. NM l 38828) are at risk of developing late-onset sporadic AD.
  • APOE Apolipoprotein E
  • This term also includes individuals with Down syndrome (DS) who invariably develop (in their third or fourth decade) cerebral amyloid (A ⁇ ) plaques and neurofibrillary tangles (NFTs), the characteristic lesions of Alzheimer disease (AD).
  • DS Down syndrome
  • a ⁇ cerebral amyloid
  • NFTs neurofibrillary tangles
  • AD Alzheimer disease
  • Recent studies have shown that the A ⁇ 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 DS 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).
  • a "neurological disorder” refers to any central nervous system (CNS) or peripheral nervous system (PNS) disease that is associated with the ⁇ -amyloidogenic processing of Amyloid Precursor Protein. This may result in neuronal or glial cell defects including but not limited to neuronal loss, neuronal degeneration, neuronal demyelination, gliosis (i.e. , astrogliosis), or neuronal or extraneuronal accumulation of aberrant proteins or toxins (e.g., amyloid- ⁇ ).
  • CNS central nervous system
  • PNS peripheral nervous system
  • CAA congophilic angiopathy
  • amyloid angiopathy This disorder is a form of angiopathy in which the same amyloid protein that is associated with Alzheimer's disease, amyloid- ⁇ (A ⁇ ), deposits in the walls of the leptomeninges and superficial cerebral cortical blood vessels of the brain. Amyloid deposition predisposes these blood vessels to failure, increasing the risk of a hemorrhagic stroke. Since it is the same amyloid protein that is associated with Alzheimer's dementia, such 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.
  • CAA cerebral hemorrhage
  • the hemorrhage within the brain is usually confined to a particular lobe and this is slightly different compared to brain hemorrhages which occur as a consequence of high blood pressure (hypertension) - a more common cause of a hemorrhagic stroke (or cerebral hemorrhage).
  • CAA is also associated with transient ischemic attacks, subarachnoid hemorrhage, Down syndrome, post irradiation necrosis, multiple sclerosis, leucoencephalopathy, spongiform encephalopathy, and dementia pugilistica.
  • patient or “patient population” refers to individual(s) diagnosed as having Alzheimer's Disease or at risk of developing Alzheimer's Disease.
  • individuals are diagnosed, or at risk of developing Familial AD.
  • the individual is diagnosed as having, or at risk of developing, Sporadic AD.
  • Diagnosis of AD may be made based on genotypic or phenotypic characteristics displayed by the individual. For example, an individual with a mutant variant of presenilin 1, presenilin 2, or APP are at risk of developing familial AD. In another, non- limiting example, individuals with the E4 variant of APOE are at risk for developing Sporadic AD.
  • Phenotypes associated with AD may be cognitive or psychiatric.
  • cognitive phenotypes include, but are not limited to, amnesia, aphasia, apraxia and agnosia.
  • psychiatric symptoms include, but are not limited to, personality changes, depression, hallucinations and delusions.
  • DSM-IV- TR Diagnostic and Statistical Manual of Mental disorders, 4th Edition (DSM-IV- TR) (published by the American Psychiatric Association) contains the following set of criteria for dementia of the Alzheimer's type:
  • the cognitive deficits are not due to other central nervous system, systemic, or substance-induced conditions that cause progressive deficits in memory and cognition;
  • NINDS-ADRDA National Institute of Neurological and Communicative Disorders and Stroke-Alzheimer's Disease and Related Disorder Association
  • A. Definite Alzheimer's disease meets the criteria for probable Alzheimer's disease and has histopathologic evidence of Alzheimer's disease via autopsy or biopsy
  • Probable Alzheimer's disease dementia established by clinical and neuropsychological examination and involves
  • Alzheimer's disease a dementia syndrome with an atypical onset, presentation, or progression and without a known etiology; any co-morbid diseases capable of producing dementia are not believed to be the cause
  • D Unlikely Alzheimer's disease: a dementia syndrome with any of the following: sudden onset, focal neurologic signs, or seizures or gait disturbance early in the course of the illness. Phenotypic manifestations of AD may also be physical, such as by the direct (imaging) or indirect (biochemical) detection of amyloid- ⁇ plaques. Quantitation of amyloid- ⁇ (1 -40) in the peripheral blood has been demonstrated using high-performance liquid chromatography coupled with tandem mass spectrometry in a linear ion trap (Du et al., J Biomol Tech. 2005;16(4):356-63).
  • ⁇ -amyloid In vivo imaging of ⁇ -amyloid can be achieved using radioiodinated flavone derivatives as imaging agents (Ono et al., J Med Chem. 2005;48(23):7253-60) and with amyloid binding dyes such as putrescein conjugated to a 40-residue radioiodinated A peptide (yielding 125 I-PUT-A 1-40), which was shown to cross the blood-brain barrier and bind to ⁇ plaques (Wengenack et al., Nature Biotechnology. 2000; 18(8): 868-72).
  • Pharmacological inhibitors of ⁇ -secretase include, but are not limited to, hydroxamic acid-based zinc metal loproteinase inhibitors, for example, batimastat. SB223820, marimastat, BB3103, BB3132, TAPI-O, TAPI-I, TAPI-2, Immunex compound 3 (IC3), KD-IX-73-4, BB21 16, and analogs thereof.
  • Batimastat also known as BB-94, is a low molecular weight synthetic inhibitor of metal loproteinase activity that functions by binding the zinc ion in the active site of MMP's.
  • the batimastat structure contains a peptide structure similar to collagen, an extracellular matrix target of the MMP's, which is bound by the MMP.
  • Batimastat also contains an hydroxamate group that binds the zinc ion in the catalytic site of the MMP, thereby inactivating it (Low, J.A., et al., Clinical Cancer Research 1996; 2: 1207-1214).
  • Batimastat, and its analogs are also inhibitors of the A Disintegrin And Metalloproteinase (ADAM) protein family, whose members ADAM9, ADAMlO, and ADAM17/TACE are candidate genes for ⁇ -secretase.
  • ADAM A Disintegrin And Metalloproteinase
  • batimastat, and its analogs function as chaperones to traffic ⁇ -secretase out of the endoplasmic reticulum
  • the ⁇ -secretase protein stabilized in a proper conformation is transported from the endoplasmic reticulum to a site of APP metabolism.
  • the pharmacological chaperone may be an inhibitor, or structurally similar analog thereof, of ⁇ -secretase.
  • inhibitors of ⁇ -secretase include, but are not limited to, the group comprising hydroxamic acid-based zinc metalloproteinase inhibitors.
  • hydroxamic acid- based zinc metalloproteinase inhibitors include, but are not limited to, the group comprising hydroxamic acid-based zinc metalloproteinase inhibitors.
  • hydroxamic acid- based zinc metalloproteinase inhibitors include hydroxamic acid- based zinc metalloproteinase inhibitors:
  • Marimastat having the structure:
  • TAPI-O having the structure:
  • TAPI-2 having the structure:
  • Immunex compound 3 having the structure:
  • BB21 16 having the structure: GM6001, having the structure:
  • rNCB8765 having the structure (l R,3S,4S)-3-[(hydroxyamino)carbonyl]-4-[(4- phenylpiperidin-l -yl)carbonyl]cyclohexyl pyrrolidine- 1 -carboxy late.
  • BB3103 is another MMP inhibitor and is available from British Biosciences.
  • Batimastat also known as BB-94, is a low molecular weight synthetic inhibitor of metalloproteinase activity that functions by binding the zinc ion in the active site of MMP's.
  • the batimastat structure contains a peptide structure similar to collagen, an extracellular matrix target of the MMP's, which is bound by the MMP.
  • Batimastat also contains an hydroxamate group that binds the zinc ion in the catalytic site of the MMP, thereby inactivating it (Low, J. A., et al., Clinical Cancer Research 1996; 2: 1207-1214).
  • Batimastat, and its analogs are also inhibitors of the A Disintegrin And Metalloproteinase (ADAM) protein family, whose members ADAM9, ADAMlO, and ADAM17/TACE are candidate genes for ⁇ -secretase.
  • ADAM A Disintegrin And Metalloproteinase
  • batimastat, and its analogs may function as chaperones to traffic ⁇ - secretase out of the endoplasmic reticulum.
  • ADAMl O inhibitors include GW280264X and GI254023X, which are described in Hundhausen et al., Blood. 2003; 102: 1 186-1 195, have the following structures:
  • ⁇ -secretase inhibitors include ADAMl O inhibitors as described in PCT publication WO03/106381 , to Bannen et al., owned by Exelixis, Inc. Such compounds are provided in the following Table:
  • TACE inhibitors include IK682, several hydroxamate-cyclopropyl compounds and series of spirocyclopropyl compounds, which are described in Guo et al., Bioorganic & Medicinal Chemistry Letters. 2009; 19: 54-57.
  • IK682 has the structure:
  • hydroxamate-cyclopropyl compounds had the structure:
  • TACE inhibitors are given in Becherer et al., book chapter "The Tumor Necrosis Factor-alpha Converting Enzyme" and are shown in the following Table:
  • EWG refers to an electron withdrawing group.
  • inhibitors disclosed are several methanesulfonyl-arginine and pyridylsulfonyl-arginine inhibitors according to the structure:
  • GI254023X having the structure:
  • the present invention also provides methods for treating a condition associated with reduced protein stability, activity, and/or trafficking, or a condition that would benefit by an increase of basal level protein stability, activity, and/or trafficking, by administering to a subject in need of such treatment a pharmacological chaperone to enhance protein stability, activity, and/or trafficking of the protein.
  • the subject to be treated can be a subject who does not exhibit a mutation in the protein that affects folding and processing of the protein, but who would benefit from increased protein stability, activity, and/or trafficking.
  • the subject to be treated can also have a mutation in the protein that affects protein function, other than a mutation that affects folding and processing of the protein, and exhibits reduced protein levels in cells that normally express the protein.
  • the subject is homozygous for the wild-type ⁇ -secretase protein. In another embodiment, the subject is heterozygous for the wild-type ⁇ -secretase protein and has a mutant genotype with a null phenotype for the other allele encoding the ⁇ -secretase protein.
  • a pharmacological chaperone i.e , an agonist or antagonist or other compound as described above or as identified through the screening methods of the invention as set forth below, is advantageously formulated in a pharmaceutical composition together with a pharmaceutically acceptable carrier.
  • the pharmacological chaperone may be designated as an active ingredient or therapeutic agent for the treatment of a disease or disorder that would benefit from an increase in protein activity, conformational stability, and/or trafficking.
  • the concentration of the active ingredient depends on the desired dosage and administration regimen, as discussed below. Exemplary dose ranges of the active ingredient are from about 0.01 mg/kg to about 250 mg/kg of body weight per day; from about 10 mg/kg to about 100 mg/kg per day; or from about 10 mg/kg to about 75 mg/kg per day.
  • Batimastat has been administered to cancer patients at doses of 600 to 1050mg/m 2 (intraperitoneal Iy).
  • Maramistat has been orally administered in pancreatic cancer patient at doses of 5, 10, or 25 mg b.i.d.
  • Therapeutically effective compounds can be provided to a subject in standard formulations, and may include any pharmaceutically acceptable additives, such as excipients, lubricants, diluents, flavorants, colorants, buffers, and disintegrants.
  • Standard formulations are well known in the art. See e.g., Remington's Pharmaceutical Sciences, 20th edition, Mack Publishing Company, 2000.
  • the formulation may be produced in useful dosage units for administration by any route that will permit the therapeutic chaperone to cross the blood- brain barrier.
  • Exemplary routes include oral, parenteral, transmucosal, intranasal, inhalation, or transdermal routes.
  • Parenteral routes include intravenous, intra-arteriolar, intramuscular, intradermal, subcutaneous, intraperitoneal, intraventricular, intrathecal, and intracranial administration.
  • a pharmacologic chaperone is formulated in a solid oral dosage form.
  • the pharmaceutical composition may take the form of a tablet or capsule prepared by conventional means with pharmaceutically acceptable excipients such as binding agents ⁇ e.g., pregelatinized maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers ⁇ e.g., lactose, microcrystalline cellulose or calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc or silica); disintegrants ⁇ e.g., potato starch or sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulphate).
  • binding agents ⁇ e.g., pregelatinized maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose
  • fillers ⁇ e.g., lactose, microcrystalline cellulose or calcium hydrogen phosphate
  • lubricants e.g., magnesium stea
  • Liquid preparations for oral administration may take the form of, for example, solutions, syrups or suspensions, or they may be presented as a dry product for constitution with water or other suitable vehicle before use.
  • Such liquid preparations may be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters, ethyl alcohol or fractionated vegetable oils); and preservatives (e.g., methyl or propyl- p-hydroxybenzoates or sorbic acid).
  • the preparations may also contain buffer salts, flavoring, coloring and sweetening agents as appropriate.
  • a pharmacological chaperone is formulated for parenteral administration.
  • the pharmacological chaperone may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion.
  • Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative.
  • the compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
  • the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.
  • the chaperone may also be formulated as a depot preparation.
  • Such long acting formulations may be administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection.
  • the compounds may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.
  • the pharmacological chaperone can be delivered in a vesicle, particularly a liposome.
  • the pharmacological chaperone can be delivered in a controlled release manner.
  • a therapeutic agent can be administered using intravenous infusion with a continuous pump, in a polymer matrix such as poly- lactic/glutamic acid (PLGA), in a pellet containing a mixture of cholesterol and the active ingredient (SilasticRTM; Dow Corning, Midland, MI; see U.S. Patent No. 5,554,601), by subcutaneous implantation, or by transdermal patch. Combination Therapy.
  • the pharmaceutical composition may also include other biologically active substances in combination with the candidate compound (pharmacological chaperone) or may be administered in combination with other biologically active substances.
  • the pharmacological chaperone can be used to treat patients with Alzheimer's Disease in combination with other drugs that are also used to treat the disorder.
  • exemplary non-limiting pharmacological agents approved in the United States for the treatment of Alzheimer's include cholinesterase inhibitors such as Cognex® (tacrine), Aricept® (donepezil), Exelon® (rivastigmine), Reminyl® (galantamine), and NMDA receptor antagonists such as Namenda® (memantine).
  • Other potential therapeutic agents include protease inhibitors (see e.g., U.S. Patent Nos. 5,863,902; 5,872,101 ; inhibitors of ⁇ -amyloid production such as described in U.S. Patent Nos.
  • the pharmacological chaperone can be used in combination with gene therapy.
  • Gene therapy is contemplated both with replacement genes such as nerve growth factor (NGF), or with inhibitory RNA (siRNA) for enzymes that are involved in the amyloidogenic cleavage of APP (BACE-I).
  • NGF nerve growth factor
  • siRNA inhibitory RNA
  • BACE-I amyloidogenic cleavage of APP
  • contemplated combination therapy includes combinations of specific pharmacological chaperones with vaccine therapy, such as described in U.S. Patent Nos. 6,866,860 and 6,761 ,888.
  • the present invention further provides a method for identifying a candidate pharmacological chaperone compound that modulates the stability, activity, and/or trafficking of an ⁇ -secretase polypeptide.
  • the present invention provides a method for identifying a chaperone for a target protein, which comprises bringing a labeled or unlabeled test compound in contact with the protein or a fragment thereof and measuring the amount of the test compound bound to the protein or to the fragment thereof. For example, this can be achieved as follows: (a) contacting a first cell with a test compound for a time period sufficient to allow the cell to respond to said contact with the test compound;
  • step (b) determining the conformational stability, activity, and/or trafficking of the target protein (or a fragment thereof comprising a ligand binding domain) in the cell (or on the cell surface) contacted in step (a);
  • step (c) comparing the conformational stability, activity, and/or trafficking of the protein determined in step (b) to that of a protein in a control cell that has not been contacted with the test compound;
  • a detectable change in the conformational stability, activity, and/or trafficking of the protein in the first cell in response to contact with the test compound compared to that same value of the protein in the control cell that has not been contacted with the test compound indicates that the test compound modulates the conformational stability, activity, and/or trafficking of the protein and is a candidate compound for the treatment of a disorder that would benefit from an increase in conformational stability, protein activity and/or proper trafficking of the target protein.
  • the ⁇ -secretase protein can either be expressed in a host cell transformed with a vector encoding a non-endogenous ⁇ -secretase protein or expressed from an endogenous gene in the cell.
  • test compounds can be, without limitation, a small organic or inorganic molecule (preferred), a peptide or a polypeptide (including an antibody, antibody fragment, or other immunospecific molecule), an oligonucleotide molecule (such as an aptamer), a polynucleotide molecule, or a chimera or derivative thereof.
  • Test compounds which are candidate pharmacological chaperones that specifically bind to an ⁇ -secretase protein can be identified using cell-based and/or cell-free assays.
  • preferred classes of compounds that may be identified include, but are not limited to/small molecules (i.e., organic or inorganic molecules which are less than about 2 kilodaltons (kD) in molecular weight, and, more preferably, less than about 1 kD in molecular weight).
  • small molecules i.e., organic or inorganic molecules which are less than about 2 kilodaltons (kD) in molecular weight, and, more preferably, less than about 1 kD in molecular weight).
  • ICCB Institute of Chemistry and Cell Biology
  • Chem Bridge DiverSet E (16,320 compounds); Bionet 1 (4,800 compounds); CEREP (4,800 compounds); Maybridge 1 (8,800 compounds); Maybridge 2 (704 compounds); Peakdale 1 (2,816 compounds); Peakdale 2 (352 compounds); ChemDiv Combilab and International (28,864 compounds); Mixed Commercial Plate 1 (352 compounds); Mixed Commercial Plate 2 (320 compounds); Mixed Commercial Plate 3 (251 compounds); Mixed Commercial Plate 4 (331 compounds); ChemBridge Microformat (50,000 compounds); Commercial Diversity Set 1 (5,056 compounds); NCI Collections: Structural Diversity Set, version 2 (1,900 compounds); Mechanistic Diversity Set (879 compounds); Open Collection 1 (90,000 compounds); Open Collection 2 (10,240 compounds); Known Bioactives Collections: NTNDS Custom Collection (1 ,040 compounds); ICCB Bioactives 1 (489 compounds); SpecPlus Collection (960 compounds); ICCB Discretes Collections.
  • ICCBl 190 compounds
  • ICCB2 352 compounds
  • ICCB3 352 compounds
  • ICCB4 352 compounds
  • Natural Product Extracts NCI Marine Extracts (352 wells); Organic fractions - NCI Plant and Fungal Extracts (1,408 wells); Philippines Plant Extracts 1 (200 wells); ICCB-ICG Diversity Oriented Synthesis (DOS) Collections; DDSl (DOS Diversity Set) (9600 wells).
  • ArQuIe, Inc. (Woburn, MA) also has integrated technologies to perform high-throughput, automated production of chemical compounds and to deliver these compounds of known structure and high purity in sufficient quantities for lead optimization. Its AMAPTM (Automated Molecular Assembly Plant) performs high-throughput chemical syntheses for each phase of compound discovery.
  • HTS screening for ⁇ -secretase protein pharmacological chaperones employs microarrays.
  • Protein arrays are solid-phase, binding assay systems using immobilized proteins on various surfaces that are selected for example from glass, membranes, microtiter wells, mass spectrometer plates, and beads or other particles.
  • the binding assays using these arrays are highly parallel and often miniaturized. Their advantages are that they are rapid, can be automated, are capable of high sensitivity, are economical in their use of reagents, and provide an abundance of data from a single experiment.
  • Automated multi-well formats are the best-developed HTS systems. Automated 96- or 384-well plate-based screening systems are the most widely used. The current trend in plate-based screening systems is to reduce the volume of the reaction wells even further, and increase the density of the wells per plate (96 wells to 384 wells to 1 ,536 wells per plate). The trend results in increased throughput, dramatically decreased bioreagent costs per compound screened, and a decrease in the number of plates that need to be managed by automation.
  • protein arrays that can be used for HTS, see e.g. : U.S. Patents No. 6,475,809; 6,406,921 ; and 6,197,599; and International Publication Nos. WO 00/04389 and WO 00/07024.
  • sources of proteins or fragments thereof can include cell-based expression systems for recombinant proteins, purification from natural sources, production in vitro by cell-free translation systems, and synthetic methods for making peptides.
  • cell-based expression systems for recombinant proteins purification from natural sources, production in vitro by cell-free translation systems, and synthetic methods for making peptides.
  • synthetic methods for making peptides For capture arrays and protein function analysis, it is often the case that ⁇ -secretase proteins are correctly folded and functional.
  • the immobilization method used is preferably applicable to proteins of different properties (e.g., wild-type, mutant, full-length, partial- length fragments, hydrophilic, hydrophobic, etc ⁇ ), amenable to high throughput and automation, and generally compatible with retention of chaperone-binding ability.
  • Both covalent or non-covalent methods of protein immobilization can be used.
  • Substrates for covalent attachment include, e.g., glass slides coated with amino- or aldehyde-containing silane reagents (Telechem).
  • VersalinxTM system Prolinx
  • reversible covalent coupling is achieved by interaction between the protein derivatized with phenyldiboronic acid, and salicylhydroxamic acid immobilized on the support surface.
  • Covalent coupling methods providing a stable linkage can be applied to a range of proteins.
  • Non-covalent binding of unmodified protein occurs within porous structures such as HydroGelTM (PerkinElmer), based on a
  • Cell-Based Arrays combine the technique of cell culture in conjunction with the use of fluidic devices for measurement of cell response to test compounds in a sample of interest, screening of samples for identifying molecules that induce a desired effect in cultured cells, and selection and identification of cell populations with novel and desired characteristics.
  • High-throughput screening can be performed on fixed cells using fluorescent-labeled antibodies, biological ligands or candidate chaperones and/or nucleic acid hybridization probes, or on live cells using multicolor fluorescent indicators and biosensors. The choice of fixed or live cell screens depends on the specific cell-based assay required.
  • Transfected cell microarrays are a complementary technique in which array features comprise clusters of cells overexpressing defined cDNAs. Complementary DNAs cloned in expression vectors are printed on microscope slides, which become living arrays after the addition of a lipid transfection reagent and adherent mammalian cells (Bailey et al., Drug Discov. Today 2002; 7(18 Suppl): Sl 13-8).
  • Cell-based arrays are described in detail in, e.g., Beske, Drug Discov. Today 2002; 7(18 Suppl): Sl 31-5; Sundberg et al., Curr. Opin. Biotechnol. 2000; 1 1 : 47-53; Johnston et al., Drug Discov. Today 2002; 7: 353-63; U.S. Patent Nos. 6,406,840 and 6,103,479, and U.S. published patent application no. 2002/0197656.
  • Detectable labels For detection of molecules such as candidate ⁇ -secretase protein pharmacological chaperones using screening assays, a functional assay can be used to follow unlabeled molecules as described elsewhere herein.
  • a molecule-of-interest ⁇ e.g., a small molecule, an antibody, or a polynucleotide probe
  • a library of same can also be detectably labeled with an atom (e.g., a radionuclide), a detectable molecule ⁇ e.g., fluorescein), or a complex that, due to a physical or chemical property, serves to indicate the presence of the molecule of interest.
  • a molecule can also be detectably labeled when it is covalently bound to a "reporter" molecule (e.g., a biomolecule such as an enzyme) that acts on a substrate to produce a detectable product.
  • reporter molecules e.g., a biomolecule such as an enzyme
  • Detectable labels suitable for use in the present invention include any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical, or chemical means.
  • Labels useful in the present invention include, but are not limited to, biotin for staining with labeled avidin or streptavidin conjugate, magnetic beads (e.g., DynabeadsTM), fluorescent dyes (e.g., fluorescein, fluorescein-isothiocyanate (FITC), Texas red, rhodamine, green fluorescent protein, enhanced green fluorescent protein, lissamine, phycoerythrin, Cy2, Cy3, Cy3.5, Cy5, Cy5.5, Cy7, FluorX from Amersham, SyBR Green I & II from Molecular Probes, and the like), radiolabels (e.g., 3 H, 125 I, 35 S, 14 C, or 32 P), enzymes (e.g., hydrolases, particularly phosphatases such as alkaline phosphatase, esterases and glycosidases, or oxidoreductases, particularly peroxidases such as horse radish peroxidase, and the like), substrates, cofactor
  • radiolabels and chemiluminescent labels can be detected using photographic film or scintillation counters; fluorescent markers can be detected using a photo-detector to detect emitted light (e.g., as in fluorescence-activated cell sorting, FACS); and enzymatic labels can be detected by providing the enzyme with a substrate and detecting, e.g., a colored reaction product produced by the action of the enzyme on the substrate.
  • lead compounds are evaluated in wild-type animals to determine whether protein activity, stability, or trafficking is increased in response to the administration of the test compound.
  • enhanced ⁇ -secretase activity, and/or cellular localization can be determined by measuring an increase in cellular polypeptide, by determining an increase in trafficking to the appropriate cellular location, and by detecting increased ⁇ -secretase protein activity.
  • Non-limiting exemplary methods for assessing each of the foregoing are described below.
  • Determining protein level Methods for determining intracellular protein levels are well-known in the art. Such methods include Western blotting, immunoprecipitation followed by Western blotting (IP Western), or immunofluorescence using a tagged protein.
  • Determining ⁇ -secretase protein trafficking and localization Assessing trafficking of proteins through the biosynthetic pathway can be achieved e.g., using pulse- chase experiments with 35 S-labeled receptor protein, in conjunction with glycosidases; or by indirect or direct immunofluorescence to determine protein modification during trafficking. These and other methods are described for example in Current Protocols in Cell Biology 2001 ; John Wiley & Sons.
  • Sensitive methods for visually detecting cellular localization also include fluorescent microscopy using fluorescent proteins or fluorescent antibodies.
  • ⁇ -secretase protein can be tagged with e.g., green fluorescent protein (GFP), cyan fluorescent protein, yellow fluorescent protein, and red fluorescent protein, followed by multicolor and time-lapse microscopy and electron microscopy to study the fate of these proteins in fixed cells and in living cells.
  • GFP green fluorescent protein
  • cyan fluorescent protein cyan fluorescent protein
  • yellow fluorescent protein yellow fluorescent protein
  • red fluorescent protein followed by multicolor and time-lapse microscopy and electron microscopy to study the fate of these proteins in fixed cells and in living cells.
  • Fluorescence correlation spectroscopy is an ultrasensitive and non-invasive detection method capable of single-molecule and real-time resolution (Vukojevic et al., Cell MoI Life Sci 2005; 62(5): 535-50).
  • SPFI single-particle fluorescence imaging
  • For localization of proteins within lipid rafts see Latif et al., Endocrinology 2003; 144(1 1): 4725-8).
  • live cell imaging see Hariguchi, Cell Struct Funct 2002; 27(5):333-4).
  • Fluorescence resonance energy transfer (FRET) microscopy is also used to study the structure and localization of proteins under physiological conditions (Periasamy, J Biomed Opt 2001 ; 6(3): 287-91).
  • less sensitive assays can be used to detect whether they are present on the membrane.
  • Such methods include immunohistochemistry of fixed cells, or whole-cell labeling using radiolabeled ligand (e.g., 125 I).
  • the next step is determining whether the candidate compound can enhance the amount of ⁇ -secretase protein trafficked to the appropriate cellular location.
  • Numerous assays can be used to evaluate protein levels quantitatively.
  • radioactive ligand binding assays using e.g., ' 5 I-MSH, can be used to determine binding to either whole cells expressing ⁇ -secretase protein or to cell membrane fractions. See U.S. published application 2003/0176425 for a description of one exemplary method; see also Chhajlani, Peptides. 1996; 17(2):349-51.
  • immunofluorescence staining using either labeled antibodies or labeled ⁇ -secretase protein ⁇ e.g., FLAG-tagged ⁇ -secretase protein
  • FACS fluorescence-activated cell sorting
  • Activity assays for proteins are generally well-known in the art, and can include substrate metabolism or modification (such as phosphorylation or dephosphorylation), signal transduction, and changes to cellular phenotype.
  • host cell means any cell of any organism that is selected, modified, transformed, grown, used, or manipulated in any way, for the production of a desired substance by the cell, for example the expression by the cell of a gene, a DNA or RNA sequence, a protein, or an enzyme. According to the present invention, the host cell is modified to express ⁇ -secretase-type nucleic acid and polypeptide. Host cells can further be used for screening or other assays. Exemplary host cells for use in the present invention are HEK-293T and COS cells.
  • a "recombinant DNA molecule” is a DNA molecule that has undergone a molecular biological manipulation.
  • Polynucleotides herein may be flanked by natural regulatory (expression control) sequences, or may be associated with heterologous sequences, including promoters, internal ribosome entry sites (IRES) and other ribosome binding site sequences, enhancers, response elements, suppressors, signal sequences, polyadenylation sequences, introns, 5'- and 3'- non- coding regions, and the like.
  • the nucleic acids may also be modified by many means known in the art.
  • Non-limiting examples of such modifications include: methylation, "caps,” substitution of one or more of the naturally occurring nucleotides with an analog, and internucleotide modifications such as, for example, those with uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoroamidates, carbamates, etc.) and with charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.).
  • uncharged linkages e.g., methyl phosphonates, phosphotriesters, phosphoroamidates, carbamates, etc.
  • charged linkages e.g., phosphorothioates, phosphorodithioates, etc.
  • Polynucleotides may contain one or more additional covalently linked moieties, such as, for example, proteins (e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), intercalators (e.g., acridine, psoralen, etc.), chelators (e.g., metals, radioactive metals, iron, oxidative metals, etc.), and alkylators.
  • the polynucleotides may be derivatized by formation of a methyl or ethyl phosphotriester or an alkyl phosphoramidate linkage.
  • the polynucleotides herein may also be modified with a label capable of providing a detectable signal, either directly or indirectly. Exemplary labels include radioisotopes, fluorescent molecules, biotin, and the like.
  • express when used in the context of producing an amino acid sequence from a nucleic acid sequence, means allowing or causing the information in a gene or DNA sequence to become manifest, for example producing a protein by activating the cellular functions involved in transcription and translation of the corresponding gene or DNA sequence.
  • a DNA sequence is expressed in or by a cell to form an "expression product", i.e., protein.
  • the expression product itself e.g., the resulting protein, may also be said to be “expressed” by the cell.
  • An expression product can be characterized as intracellular, extracellular or secreted.
  • heterologous refers to a combination of elements not naturally occurring in combination.
  • heterologous DNA refers to DNA not naturally located in the cell, or in a chromosomal site of the cell.
  • the heterologous DNA includes a gene foreign to the cell.
  • a heterologous expression regulatory element is an element operatively associated with a different gene than the one it is operatively associated with in nature.
  • a gene encoding a protein of interest is heterologous to the vector DNA in which it is inserted for cloning or expression, and it is heterologous to a host cell containing such a vector, in which it is expressed, e.g., an E. coli cell.
  • recombinantly engineered cell refers to any prokaryotic or eukaryotic cell that has been manipulated to express or overexpress the nucleic acid of interest, i.e., a nucleic acid encoding a polypeptide, by any appropriate method, including transfection, transformation or transduction. This term also includes endogenous activation of a nucleic acid in a cell that does not normally express that gene product or that expresses the gene product at a sub-optimal level.
  • vector means the vehicle by which a DNA or RNA sequence can be introduced into a host cell, so as to transform the host and promote expression (e.g., transcription and translation) of the introduced sequence.
  • Vectors include plasmids, phages, viruses, etc.; they are well known in the art.
  • This example describes a randomized, blinded, Phase Ib study of twice daily oral doses of 1-deoxygalactonojirimycin (DGJ) to evaluate the affects of DGJ on safety, tolerability, pharmacokinetics, and ⁇ -Galatosidase A ( ⁇ -GAL) enzymantic activity in healthy volunteers. It demonstrates the power of the pharmacological chaperone approach to increase the levels and/or activity of a wild-type enzyme in the normal, healthy subject.
  • DGJ 1-deoxygalactonojirimycin
  • Study Design and Duration This study was first-in-man, single-center, Phase Ib, randomized, double-blind, twice daily-dose, placebo controlled study to evaluate the safety, tolerability, pharmacokinetics, and ⁇ -GAL enzymantic activity affects of DGJ following oral administration.
  • the study tested two groups of of 8 subjects (6 active and 2 placebo) who received a twice daily-dose of 50 or 150 mg b.i.d. of DGJ or placebo administered orally for seven consecutive days, accompanied by a seven day follow up visit. Subjects were housed in the treatment facility from 14 hours prior to dosing until 24 hours after dosing. Meals were controlled by schedule and subjects remained abulatory for 4 hours post drug administration
  • Safety and Tolerability Assessments were determined by evaluating vital signs, laboratory parameters (serum chemistry, hematology, and urinalysis), ECGs, physical examination and by recording adverse events during the Treatment Period.
  • WBC a-GAL A Enzymatic Activity Sampling Blood samples (10 itiL each) were collected in blood collection tubes containing EDTA and WBC extracted before dosing and at the following times thereafter: 100 hours, 150 hours, and 336 hours. Samples were treated as described above, and WBC ⁇ -GAL enzymatic activity levels were determined as described in Desnick, R.J. (ed) Enzyme therapy in genetic diseases. VoI 2. Alan R Liss, New York, pp 17-32. Statistical Analysis. Safety data including laboratory evaluations, physical exams, adverse events, ECG monitoring and vital signs assessments were summarized by treatment group and point of time of collection.
  • Descriptive statistics (arithmetic mean, standard deviation, median, minimum and maximum) were calculated for quantitative safety data as well as for the difference to baseline. Frequency counts were compiled for classification of qualitative safety data.
  • a shift table describing out of normal range shifts was provided for clinical laboratory results.
  • a normal-abnormal shift table was also presented for physical exam results and ECGs.
  • Adverse events were coded using the MedDRA version 7.0 dictionary and summarized by treatment for the number of subjects reporting the adverse event and the number of adverse events reported. A by-subject adverse event data listing including verbatim term, coded term, treatment group, severity, and relationship to treatment was provided. Concomitant medications and medical history were listed by treatment.
  • Pharmacokinetic parameters were summarised by treatment group using descriptive statistics (arithmetic means, standard deviations, coefficients of variation, sample size, minimum, maximum and median).
  • C m3x of DGJ increased in a dose-proportional manner when doses were increased from 50 mg to 150 mg.
  • the mean percentage of DGJ excreted over the 12-hour post dose period was 16% and 42% at dose levels of 50 and 150 mg, respectively, on Day 1, increasing to 48% and 60%, respectively, on Day 7.
  • a-Galactosidase A (a-Gal A) Enzymatic Activity.
  • the ⁇ -GAL enzymatic activity data obtained during the study is shown in Figure 1.
  • DGJ did not inhibit WBC ⁇ -GAL enzymatic activity in subjects at dosages of 50 mg b.i.d. or 150 mg b.i.d.
  • AT- 1001 produced a dose-dependent trend of increased WBC ⁇ -GAL activity in healthy volunteers.
  • ⁇ -GAL enzymatic levels were measured in WBC of subjects administered placebo, 50 mg b.i.d. DGJ, and 150 mg b.i.d. DGJ. Placebo had no affect on WBC ⁇ -GAL enzymatic levels.
  • Blood samples were collected for pharmacokinetic analysis before the initial drug administration on Day 1 , before the 5th, 6th and 7th doses (on Days 5, 6 and 7) (for Cmin determination), and at the following times after the 1st (Day 1 ) and 7th (Day 7) doses: 0.5, 1 , 1.5, 2, 3, 4, 5, 6, 8, 10, 12, 15, 18, and 24 hours.
  • a single blood sample was collected 48 hours after the last dose (Day 9) and assayed for the presence of IFG.
  • blood samples were collected for pharmacodynamic measurements, i.e., analysis of WBC GCase levels, before dosing on Day 1 , Day 3, Day 5, and Day 7, and at return visits on Day 9, Day 14 and Day 21.
  • Patients were administered DGJ according to the following dosing schedule: 25 mg b.i.d. two weeks; 100 mg b.i.d. 2-4 weeks; 250 mg b.i.d. 4-6 weeks; and 25 mg b.i.d. 6-12 weeks; blood was draw into an 8 mL Vacutainer CPT tube at the end of each dosing period and treated as described below.
  • WBCs were prepared substantially as described in Example 1 , with the exception that no FBS/DMSO is added to the pellet prior to freezing.
  • lysis buffer 26 mM citrate/46 mM phosphate, pH 5.5
  • Tubes were incubated at room temperature for about 15 minutes, but agitate the suspension by vortexing every couple of minutes
  • Tubes were sonicated for 2 minutes, then vortexed for about 10 seconds
  • Container was vortexed and pooled lysates were divided into 0.100 mL aliquots in pre-chilled labeled 0.5 mL screw-cap polypropylene microcentrifuge tubes. Pooled lysates were mixed while aliquoting by vortexing between every 10-20 aliquots.
  • EXAMPLE 4 In vitro Stabilization of purified ADAMlO by TAPI-2, GM6001 and GW4023
  • Figure 3 depicts a time course (96 hr) showing the effects of GM6001 (Figure 3A) and TAPI-2 (Figure 3B) on ADAMlO levels in SY5Y neuroblastomas.
  • Figure 3C depicts a dose-response showing the effects of TAPI-2 on ADAMlO precursor levels in SY5Y neuroblastomas.
  • ADAMlO has a T m of 52 0 C.
  • the ADAMlO inhibitors TAPI-2 and GW4023 shift the T n , by 9 0 C and 16 0 C, respectively, demonstrating their ability to stabilize wilt-type ADAMlO.
  • SY5Y neuroblastoma cells in 6-well plates were dosed with lOOuM of alpha secretase inhibitors GM6001 (AA) or TAPI-2 (AB) in complete growth media (DMEM+10%FBS) for up to 96hrs.
  • AA alpha secretase inhibitor
  • TAPI-2 TAPI-2
  • DMEM+10%FBS complete growth media
  • Ohrs no dose control
  • 15hrs, 24hrs, 48hrs, 72hrs and 96hrs cells were harvested by centrifugation and lysed in 75ul of ice cold 2%CHAPS/TBS + protease inhibitors. Lysates were quantified by BCA (Pierce Chem.
  • ADAMlO was probed using a rabbit anti- ADAMlO polyclonal antibody (1 : 1000, Abeam cat. No. Abl997) and anti-rabbit/alkaline- phosphatase secondary antibody.
  • ADAMl O bands were imaged using an alkaline phosphatase chemiluminescence substrate (CDP-Star, Invitrogen) and FluorChem Q digital imager (Alpha Innotech). Densitometry quantification was done using AlphaView Q software (Alpha Innotech).
  • SY5Y neuroblastoma cells in 6-well plates were dosed with OuM, 0.01 uM, 0. IuM, IuM, l OuM and lOOuM of the alpha secretase inhibitor TAPI-2 in complete growth medium (DMEM+10% FBS). After 96hrs, cells were harvested by centrifugation and lysed in 75ul of ice cold 2%CHAPS/TBS + protease inhibitors. Lysates were quantified by BCA (Pierce Chem. Co.) and equal amounts of protein were electrophoresed on a 4-12% Bis-NuPage gel (Invitrogen) followed by transfer (25V, l hr) onto PVDF for Western blotting.
  • ADAM lO was probed using a rabbit anti-ADAMIO polyclonal antibody (1 : 1000, Abeam cat. No. Ab 1997) and anti-rabbit/alkaline-phosphatase secondary antibody.
  • ADAMlO bands were imaged using an alkaline phosphatase chemiluminescence substrate (CDP-Star, Invitrogen) and FluorChem Q digital imager (Alpha Innotech). Densitometry quantification was done using AlphaView Q software (Alpha Innotech).
  • the environmentally sensitive dye SYPRO Orange and a Realplex Mastercycler qRT- PCR system were used to apply a temperature gradient while simultaneously monitoring fluorescence changes in a 96-well format.
  • ADAMlO in neutral buffer (50 mM sodium phosphate, 150 mM sodium chloride, 2 ⁇ M zinc chloride, pH 7.4) was combined with 2.5 ⁇ l 50X SYPRO Orange and 20 ⁇ M TAPI-2 in a reaction volume of 25 ml. Once assembled, the plate is heated at a rate of l°C/min, and fluorescence (Ex. 470 nm; Em. 520 nm) intensities are measured and plotted as a function of temperature.
  • ADAMl O demonstrates a typical thermal denaturation curve with a T m (melting temperature) of 52 0 C.
  • T m melting temperature
  • the T m is shifted to 61 0 C, a shift of 9 0 C (Fig. 2A), while another more potent inhibitor GW4023 shifted the T m by 16 0 C (data not shown).
  • GW4023 is also known as GI254023X

Abstract

L’invention concerne des procédés d’augmentation de l’expression de α-sécrétase normale, et d’augmentation de l’expression d’une α-sécrétase ayant une mutation qui affecte le repliement et/ou la transformation de protéine de la α-sécrétase. L’invention concerne un procédé de traitement d’un individu ayant une affection dans laquelle l’expression ou l’activité augmentée d’une α-sécrétase dans le système nerveux central serait bénéfique, par administration d’une quantité efficace d’un chaperon pharmacologique pour la α-sécrétase. L’invention concerne des inhibiteurs de α-sécrétase qui ont été identifiés en tant que chaperons pharmacologiques spécifiques qui augmentent l’expression ou l’activité enzymatique de α-sécrétase dans le système nerveux central.
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EP2490712A1 (fr) * 2009-10-19 2012-08-29 Amicus Therapeutics, Inc. Méthode de traitement de la maladie d'alzheimer au moyen de chaperons pharmacologiques destinés à augmenter l'activité des gangliosidases
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EP2490712A1 (fr) * 2009-10-19 2012-08-29 Amicus Therapeutics, Inc. Méthode de traitement de la maladie d'alzheimer au moyen de chaperons pharmacologiques destinés à augmenter l'activité des gangliosidases
EP2490712A4 (fr) * 2009-10-19 2013-06-12 Amicus Therapeutics Inc Méthode de traitement de la maladie d'alzheimer au moyen de chaperons pharmacologiques destinés à augmenter l'activité des gangliosidases
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EP2957295A1 (fr) * 2009-10-19 2015-12-23 Amicus Therapeutics, Inc. Procédé de traitement de la maladie d'alzheimer utilisant des chaperons pharmacologiques pour augmenter l'activité des gangliosidases
US9931353B2 (en) 2009-10-19 2018-04-03 Amicus Therapeutics, Inc. Method for treating cerebral amyloid angiopathy using pharmacological chaperones to increase the activity of gangliosidases
WO2011076854A1 (fr) * 2009-12-22 2011-06-30 Probiodrug Ag Clivage de protéine précurseur de β-amyloïde
US9675627B2 (en) 2014-04-14 2017-06-13 Amicus Therapeutics, Inc. Dosing regimens for treating and/or preventing cerebral amyloidoses

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