Protein activator and apoptosis

Details Protein activator and apoptosis Protein activator and apoptosis

2000-03-21

2001-07-31

Schwartzman, Robert A. (Department: 1635)

Drug, bio-affecting and body treating compositions

Enzyme or coenzyme containing

C435S183000, C530S350000

Reexamination Certificate

active

06267956

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to nucleic acid and amino acid sequences pertaining to a kinase of death (KOD) biomolecule which is integral to the activation process of cellular apoptosis. Molecular sequences are provided for the design and synthesis of entities that modulate biological and/or pharmacological activity of the native biomolecule. The sequences are also provided for employment to identify compounds that modulate biological and/or pharmacological activity of the native biomolecule. Biologically-effective antisense molecules are provided, as well as dominant negative mutant versions of the kinase of death which are suitable for therapeutic use. The invention is also drawn toward the study, prevention, diagnosis, and treatment of pathophysiological disorders related to apoptosis.
BACKGROUND OF THE INVENTION
Apoptosis, or programmed death at the level of the cell, is needed to orchestrate biological maintenance of the organism during development as well to preserve the normal function and fitness of tissues during a normal life span. Physiological conditions which result from aberrant apoptosis may be dire. Cancer and autoimmune disease may result when there is too little apoptosis, as well as severe stroke damage or the neurodegeneration of Alzheimer's disease when there is too much. Miller, L. J.,
Apoptosis, Science
, 281:1 (1998). Apoptotic neuronal cell death appears to be a common denominator of a number of common neurological and psychiatric disorders. Anderson, A. J., et al., A Potential Role for Apoptosis in Neurodegeneration and Alzheimer's Disease, Mol. Neurobiol., 10:19 (1995); W. G. Tatton, et al., Apoptosis in Neurodegenerative Disorders: Potential for Therapy by Modifying Gene Transcription, J. Neural Transm., 49:245 (1997). CNS disorders as well as other disorders wherein apoptosis is believed to contribute to the pathogenesis include the likes of stroke, ischemic injury, Alzheimer's disease, Parkinson's disease, Huntington's Chorea, Amyotrophic Lateral Sclerosis (ALS), hereditary retinal degenerations, glaucoma, cachexia, and spinal muscular atrophy. Apoptosis of glial cells may also contribute to disorders like multiple sclerosis and diabetic peripheral neuropathy.
Apoptosis is known to be involved in the course of a wide variety of developmental processes including normal physiological maturation of the human immune as well as nervous systems. Normal physiological signals activate apoptosis in the context of orderly development in various tissues. Glucocorticoids, withdrawal of cytokines, DNA damage and signals through the antigen receptors of T or B lymphocytes can all induce apoptosis, depending on the inherent susceptibility of each individual cell type. Apoptosis, moreover, has been implicated as an important cellular safeguard against tumorigenesis. Irradiation and chemotherapy are also used artificially to effectively trigger apoptosis for therapeutic purposes. Expression of the natural p53 tumor suppresser gene is required for efficient induction of apoptosis following DNA damage and other physiological damage to the cell. In fact, the cytotoxicity of many commonly used chemotherapeutic agents is mediated by wild-type p53. Moreover, the p53 tumor suppresser gene is frequently mutated in human tumors. The loss of p53 function, and hence compromised apoptosis, may contribute to the clinically significant problem of drug resistant tumor cells emerging following chemotherapy regimens.
Cells have surface sensors termed death receptors which detect and respond to the presence of extracellular death signals by triggering intrinsic apoptosis machinery. These receptors can activate death caspases and within seconds of ligand binding cause an apoptotic demise of the cell. Death receptors belong to the tumor necrosis factor (TNF) receptor gene superfamily, which is defined by similar, cysteine-rich extra cellular domains. The death receptors contain in addition a homologous cytoplasmic sequence termed the “death domain”. Death domains typically enable death receptors to engage the cell's apoptotic machinery, but in some instances they mediate functions that are distinct from or even counteract apoptosis. Some molecules that transmit signals from death receptors contain death domains themselves. Ashkenazi, Avi, et al., Death Receptors: Signaling and Modulation, Science, 281:13 (1998). Death receptors have been identified on the surface of cells including the tumor necrosis factor receptor 1 (TNF-R1) and Fas receptor (FasR). The idea of targeting specific death receptors to induce apoptosis in tumors is attractive, because death receptors have direct access to the caspase machinery. Moreover, unlike many chemotherapeutic agents or radiation therapy, death receptors initiate apoptosis independently of the p53 tumor suppressor gene, which is inactivated by mutation in more than half of human cancers. Id.
Cell death via apoptosis is mediated by cysteine proteases, caspases, protein-clipping enzymes, which cleave specifically at aspartic acid, to orchestrate the cell's death program. Once the cascade of caspases have been activated, a series of degradative proteolytic events occur in the cell. In drug-resistant cells, apoptosis fails because of defects in signaling pathways that lead to caspase activation. Therapeutic opportunities are expected to exist by means of bypassing these defects. Death receptors may be activated, for example, resulting in activation of a corresponding initiator caspase. Thornberry, N. A., Caspases: Enemies Within, Science, 281:1312 (1998).
Ligation of the extracellular domain of the cell surface receptor Fas/APO-1 (CD95) elicits a characteristic apoptotic programmed death response in susceptible cells. Stanger, B. Z., et al., RIP: A Novel Protein Containing a Death Domain That Interacts with Fas/APO-1 (CD95) in Yeast and Causes Cell Death, Cell, 81:513 (1995). Stanger et al have identified two gene products that associate with the intracellular domain of Fas: Fas itself, and a 74 Kda protein Receptor Interacting Protein (RIP). RIP contains an N-terminal region with homology to protein kinases and a C-terminal region containing a cytoplasmic motif (death domain) present in the Fas and TNFR1 intracellular domains. Transient overexpression of RIP causes transfected cells to undergo the morphological changes characteristic of apoptosis. Furthermore, the death domain serine/threonine kinase RIP has been demonstrated to interact with the death receptors Fas and tumor necrosis receptor 1 (TNFR1). In vitro, RIP stimulates apoptosis, as well as SAPK/JNK and NF-kB activation. Kelliher, M. A., et al., The Death Domain Kinase RIP Mediates the TNF-Induced NF-kB Signal, Immunity, 8:297 (1998).
Entities which are able to affect the activity of specific biological molecules which activate apoptosis are expected to have significant potential for the ability to control apoptosis as well as disease conditions related thereto.
SUMMARY OF THE INVENTION
The present invention is directed to an isolated and purified polynucleotide molecule comprising a nucleic acid sequence which encodes a polypeptide comprising the sequence as depicted in SEQ ID NO:3 or a variant of SEQ ID NO:3 having at least about 80% homology to a member selected from the group consisting essentially of: (SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8).
Isolated and purified polynucleotides of the present invention include but are not limited to sequences comprising SEQ ID NO:1 and SEQ ID NO:2.
The current invention is directed to a purified polypeptide comprising the amino acid sequence as depicted in SEQ ID NO:3 or a variant thereof as defined herein.
A preferred embodiment of the invention is an isolated and purified biologically effective antisense polynucleotide molecule comprising an oligomer in the range from about 12 to about 25 nucleotides in length which is complementary to a region within positions about 150-230 of SEQ ID NO:1.
The present invention is also directed to an isolated and purified polynucleotide

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