Assays and kits for inhibition of polyglutamine-induced cell...

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Reexamination Certificate

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C435S004000, C435S183000

Reexamination Certificate

active

06177259

ABSTRACT:

FIELD OF THE INVENTION
The invention relates in general to the screening of candidate pharmacological compositions.
BACKGROUND OF THE INVENTION
Expanded tracts of CAG trinucleotide repeats, encoding polyglutamine, are now known to be the cause of several neurodegenerative diseases, among which are Huntington's Disease (HD; Huntington's Disease Collaborative Research Group, 1993,
Cell,
72: 971-983) and several types of ataxia, including spinal and bulbar muscular atrophy (La Spada et al., 1991,
Nature Genetics,
6: 14-18), dentatorubral-pallidoluysian atrophy (Koide, 1994,
Nature Genetics,
6: 9-13; Nagafachi et al., 1994,
Nature Genetics
, 6: 14-18), and five dominantly inherited ataxias-spinocerebellar ataxia (SCA) types 1-3 (Imbert et al., 1996,
Nature Genetics
, 14: 285-291; Kawaguchi et al., 1994,
Nature Genetics,
8: 221-228; Orr et al., 1993,
Nature Genetics
, 4: 221-226; Pulst et al., 1996,
Nature Genetics
, 14: 269-276; Sanpei et al., 1996,
Nature Genetics
, 14: 277-284), 6 (Zhuchenko et al., 1997,
Nature Genetics
, 15: 62-69) and 7 (David et al., 1997,
Nature Genetics
, 17: 65-70). These devastating autosomal dominant inherited disorders are characterized by impaired motor control and, in some cases, varying degrees of psychatric and cognitive deficiency (Huntington G., 1872, “On chorea”,
Med. Surg. Rep.,
26: 317-321). Expansion of the glutamine-encoding sequence appears to result in a toxic gain of function that is selectively deleterous to the neurons affected in these diseases (Paulson and Fischbeck, 1996,
Ann Rev. Neurosci
., 19: 79-107). The genes which are linked to the risk of such neurological disease encode proteins that share no significant sequence homology with each other with the exception of the abnormally long polyglutamine regions.
Expression of truncated cDNAs encoding mostly expanded CAG repeats has been shown to induce cell death, but this effect is not seen when full-length proteins comprising the repeats are produced (Davies et al., 1997,
Cell
, 90: 537-548; Ikeda et al., 1996,
Nature Genetics
, 13: 196-202); therefore, it has been hypothesized that a truncated protein fragment derived from the full-length proteins in each of these disorders may be responsible for the as yet unidentified toxic gain of function leading to the degeneration of different neuronal populations characteristic of each disorder.
Defects in the gene encoding the protein Huntingtin are linked to the development of HD.
Expression of an allele of exon 1 of this gene, which allele contained greater than 21 CAG repeats, has been found to be sufficient to induce pathogenesis and clinical symptoms resembling those of HD in transgenic mice (Bates and Davies, 1997,
Mol. Med. Today,
3: 508-515). The proteins with expanded polyglutamine repeats have been observed to form aggregates or cytoplasmic and nuclear inclusions in cultured cells overexpressing a truncated MJD protein with an expanded polyglutamine (Ikeda et al., 1996, supra), in transgenic mice expressing a truncated Huntingtin (Bates and Davies, 1997, supra), Drosophila expressing a truncated human ataxin-3 (Warrick et al., 1998,
Cell,
93: 939-949), and in postmortem SCA 3 patient brain (Paulson et al., 1997,
Neuron
, 19: 333-344); in non-human models, such aggregates and inclusions resemble those observed in sectioned brain tissue derived from affected humans. It has been proposed that such abnormal inclusions may engage in inappropriate protein-protein interactions which lead to cell death. Neither the timing of formation of such inclusions (i.e., whether it preceeds or follows induction of the death program) nor the nature of any causal role for these structures in neurotoxicity has as yet been determined.
There is need in the art for methods and reagents directed at the inhibition of polyglutamine-mediated cell death.
SUMMARY OF THE INVENTION
The invention provides a method of screening for an inhibitor of cell death, comprising an assay comprising the steps of incubating a first protein comprising a caspase, a second protein comprising a polyglutamine stretch and a candidate inhibitor of cell death under conditions sufficient to permit the formation of a complex comprising the first protein and the second protein, and performing a detection step to detect the formation of a complex, wherein a reduction in the formation of a complex in comparison to that observed when the first and second proteins are incubated in the absence of the candidate inhibitor indicates that the candidate inhibitor is an inhibitor of cell death.
As used herein in reference to cell death, the term “inhibitor” refers to an agent which is effective to mediate a reduction in the rate of polyglutamine-inclusion-induced cell death. The efficacy of a candidate inhibitor screened according to the invention may be judged by direct observation of cell survival or, alternatively, of the recruitment of a caspase to a polyglutamine molecule, aggregate or inclusion within a tissue, cell or cell-free assay system of the invention or disruption of protein:protein binding.
As used herein in reference to the action of an inhibitor identified according to the invention, the term “reduction” refers to a decrease of at least 10%, preferably 20-50%, more preferably 75-90% and, most preferably, 95-100% in the mortality of cells having polyglutamine inclusions or aggregates or in the recruitment of a caspase to polyglutamine molecules, inclusions or aggregates.
As used herein with regard to an inhibitor of cell death, the term “candidate” refers to an agent which is assayed according to the invention for efficacy in inhibiting polyglutamine-inclusion-induced cell death; such an agent may be a protein, nucleic acid or other substance, organic or inorganic, as described hereinbelow.
As used herein, the term “caspase” refers to a protein of the caspase protein family including, but not limited to, caspases 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12, proteins which encompass the sequence of—or are substantially similar (e.g. ≧50% identical in amino acid sequence) to a caspase protein family member or a peptide fragment thereof which comprises sequence of such a protein. A caspase of the invention may be mutant or wild-type, and may additionally be a recombinant protein, including a fusion protein.
As used herein, the term “polyglutamine stretch” refers to an amino acid sequence which is not normally present in a protein, and comprising a repeated (or expanded) number of glutamine residues. Such a stretch may comprise all or part of a protein, peptide or fragment thereof and, as described below, occurs in many different proteins. A polyglutamine stretch is defined as comprising 36 or more glutamine residues; typically, polyglutamine sequences associated with cell death are at least 36, and preferably more, such as 36, 46, 56, 66, 79, 109, 218, 436 or even up to 1000 glutamine residues in length. A particularly preferred polyglutamine of use in the assays of the invention comprises 79 glutamine residues; it is herein referred to as glutamine
79
or Q79.
Preferably, the protein comprising a caspase is selected from the group that includes caspases 1,2,3,4,5,6,7,8,9,10,11 and 12.
It is preferred that the polyglutamine repeat is glutamine
79
(Q79).
In another preferred embodiment, in the step of incubating the first and second proteins, an adaptor protein is incubated with the first and second proteins.
As used herein, the term “adaptor protein” refers to a protein which permits binding of a first and second protein, either by modifying or interacting with (e.g., binding) the first or second protein such that it is then able to bind the other directly, or by binding both of the first and second proteins, thereby creating a bridge such that the first and second proteins are present in the same protein complex but do not contact each other directly.
Preferably, the adaptor protein comprises a death domain (DD) and one of a death effector domain (DED) and a caspase-recruitment domain (CASP).
As used herein, the term “death domain” refers to an ami

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