Chemistry: natural resins or derivatives; peptides or proteins; – Peptides of 3 to 100 amino acid residues – 6 to 7 amino acid residues in defined sequence
Reexamination Certificate
2001-07-09
2004-08-24
Chernyshev, Olga N. (Department: 1646)
Chemistry: natural resins or derivatives; peptides or proteins;
Peptides of 3 to 100 amino acid residues
6 to 7 amino acid residues in defined sequence
C514S002600, C514S016700
Reexamination Certificate
active
06780971
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to drug screening methods and methods of preventing neural tissue damage caused by &agr;-synuclein aggregation. These methods are especially useful in the design and development of inhibitors of Lewy body diseases and other synucleinopathies, and further useful in the treatment of such neurodegenerative diseases, particularly Parkinson's Disease.
BACKGROUND OF THE INVENTION
Parkinson's disease (PD) is a common neurodegenerative disorder and was first described by James Parkinson in 1817. The four primary diagnostic signs of the illness are resting tremor, bradykinesia, muscular rigidity and postural instability. These signs of motor deficiency result from the loss of dopaminergic neurons in the nigrostriatal system [Gibb, W., et al.,
J. Neurol. Neurosurg. and Psych
., 51:745-52 (1988)].
PD is characterized by the formation of Lewy bodies and death of dopaminergic neurons. [Adams D. et al.,
Principles of Neurology
, 874-880, 3rd Edition, McGraw-Hill, N.Y., (1985)]. The neuropathological hallmark of PD is the Lewy body. Lewy bodies are intracytoplasmic inclusions that occur in degenerating neurons which are composed of a dense core of filamentous and granular material surrounded by radically oriented filaments that have a diameter of 10-20 nm [Goedert, 20 M., et al.,
Curr. Op. Neurobio.
8:619-32 (1999)]. In general, the causes of PD are not known and there has been vigorous debate over the relative roles of genetics and environmental factors [Tanner, C., et al.,
JAMA
, 281:341-6 (1999)]. Exposure to manganese precipitated a Parkinsonian syndrome in miners which also includes schizophrenia form behaviors. Some epidemiological studies have found an association between industrial exposure to iron and the incidence of PD [Corell J. M et al.,
Toxicol. Appl. Pharmacol
., 80:467-72, (1985)], between incidence of PD and blood mercury levels [Ngim C. H. et al.,
Neuroepi
., 8(3):128-141 (1989)] and with death rates from PD and proximity to iron related industrial processed [Rybicki A. et al.,
Mov Disord
., .8(1):87-92_(1993)].
&agr;-Synuclein was originally identified as a protein that is upregulated associated with neuron outgrowth during the critical period of Zebra finch song learning [George M., et al.,
Neuron
, 15:361 (1995)]. &agr;-Synuclein is a ubiquitous protein that shares significant physical and functional homology to the protein chaperone, 14-3—3, and is particularly abundant in the brain (Ostrerova N. et al.,
J. Neurosci
., 19:5782 (1990); Clayton D. et al., TINS 21:249 (1998)]. &agr;-Synuclein is normally phosphorylated at serines 87 and 129. (Okochi M. et al.,
J. Biol. Chem.,
275:390 (2000)]. Recent studies showed that mutations in &agr;-synuclein can cause familial PD and that &agr;-synuclein accumulates in Lewy bodies. These discoveries suggest that &agr;-synuclein participates in the pathophysiology of PD. (Spillantini M. et al.,
Nature
, 388:839 (1997); Spillantini M. et al.,
PNAS USA
, 95:6469 (1998); Jenner P. et al.,
Ann. Neurol
., 44:S72 (1998)]. The only identified mutations associated with familial PD to date are the A53T and A30P mutations in the &agr;-synuclein gene (Goedert, M., et al.,
Curr. Op. Neurobio
., 8:619-32 (1999); Papadimitriou, A., et al.,
Neurology
, 52:651-4 (1999); Polymeropoulos, M., et al.,
Science
, 276:1197-9 (1997)]. However, there has been much circumstantial evidence implicating oxidative stress in the etiology of the disease (Jenner, P., et al.,
Annual Neurol.,
44:S72-84 (1998)].
A variety of experimental evidence suggests that Lewy bodies interact with &agr;-synuclein. For example, immunohistochemical studies indicate that Lewy bodies stain strongly for &agr;-synuclein and ubiquitin (Jenner, P., et al.,
Annual Neurol
., 44:S72-84_(1998); Markopoulou, K., et al.,
Annual. Neurol
., 46:374-81 (1999); Spillantini, M., et al.,
Nature
, 388:839-40 (1997); and Spillantini, M, et al.,
Proc. Natl. Acad. Sci. USA
, 95:6469-73 (1998)]. In vitro experiments using recombinant protein suggest that the mutations, A53T and A30P, increase &agr;-synuclein aggregation in comparison with the wild type &agr;-synuclein (Conway, K., et al.,
Nature Med
., 4:1318-20 (1998); Giasson, B., et al.,
J. Biol. Chem
., 274:7619-22 (1999); Hashimoto, M., et al.,
Brain Res
., 25 799:301-6 (1998)].
One of the important questions regarding &agr;-synuclein aggregation and Lewy body formation is whether these processes harm the cell. Lewy bodies could either be inert tombstone markers that occur in response to free radical damage, or they might be toxic agents that harm the cell. Examples of both situations exist in the literature. Aggregated amyloid-&bgr; (A&bgr;) is toxic to neurons, while lipofuscin appears to be innocuous to cells (Behl, C., et al., Cell 77:817-27 (1994)]. The Huntington's protein presents an intermediate situation where the toxicity associated with Huntington's appears to precede aggregation, and aggregation of Huntington's protein might even be protective [Saudou, F., et al., Cell 95:55-66 (1998)]. Our own previous studies showed that transient over-expression of &agr;-synuclein is toxic to a variety of cells, including two neuronal cell lines, SK-N-SH and PC12 [Ostrerova, N., et al.,
Neurosci
., 19:5782-91 (1999)]. Consistent with this observation, Masliah and colleagues have recently shown that mice over-expressing &agr;-synuclein show an age-related loss of dopaminergic terminals and motor impairment, which could be indicative of toxicity [Masliah, E. et al.,
Science
, 287:1265-1269 (2000)]. These findings suggest that an increased rate of &agr;-synuclein aggregation might contribute to the mechanisms of neurodegeneration in PD and other Lewy body diseases.
Recent studies on transgenic animals also suggest that aggregation of &agr;-synuclein is harmful to neurons. It was recently reported that dopaminergic dysfunction occurred in transgenic mice expressing wild type human &agr;-synuclein [Masliah, E., et at.,
Science
, 287:1265-1269 (2000)]. Further, it was reported that Drosophila over-expressing &agr;-synuclein exhibited dopaminergic dysfunction and dopaminergic neuronal death associated with development of &agr;-synuclein aggregates [Feany, M B, et al.,
Nature
404:394-8 (2000)]. Evidence suggests that neurons with dopamine develop &agr;-synuclein aggregates and degenerate as these aggregates development.
Recently, oxidative stress produced by iron and hydrogen peroxide has been shown to induce amyloid-like aggregate formation of &agr;-synuclein in vitro [Hashimoto, M., et at.,
NeuroReport
, 10:717-21 (1999); Paik, S., et al.,
Biochem. J
., 340:821-8 (1999)]. Oxidative stress is thought to contribute to PD because dopamine, which is a strong free radical generator, is the principle neurotransmitter in the substantia nigra [Chiueh, C., et at.,
Adv. Neurol
., 60:251-8 (1993); Jenner, P. et al.,
Ann. Neurol
., 25 44:S72-84 (1998)]. In addition, iron, which also stimulates free radical production, accumulates in the substantia nigra with age [Jenner, P., et al.,
Ann. Neurol
., 44:S72-84 (1998)]. Iron is deposited as hemosiderin granules in the cytoplasm, and mitochondria filled with ferritin granules have been observed in the neuronal and glial cells of the ventorlateral thalamus, caudate and lenticular nuclei and substantia nigra of Parkinsonian brains. [Earle M.,
J. Neuropathol. Exper. Neurol
., 27(1):1-14, (1968); Asenjo A. et al.,
Rev. Neurologique
, 121 (6):581-92, (1969); Riederer P., et al.,
J. Neurochem
., 52(2):515-20, (1989)]. Thus, the oxidative conditions present in the substantia nigra could promote &agr;-synuclein aggregation. However, in the prior art, whether such oxidative conditions actually promote a-synuclein aggregation in living neurons is unknown.
A need, therefore, exists for substances that will inhibit
Lebowitz Michael S.
Ostretova-Golts Natalie
Wolozin Benjamin
Butler, Esq. Gregory B.
Chernyshev Olga N.
Corless, Esq. Peter F.
Edwards & Angell LLP
Panacea Pharmaceuticals, Inc.
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