Chemistry: natural resins or derivatives; peptides or proteins; – Peptides of 3 to 100 amino acid residues – 25 or more amino acid residues in defined sequence
Reexamination Certificate
1999-07-23
2001-12-04
G., Anita (Department: 1642)
Chemistry: natural resins or derivatives; peptides or proteins;
Peptides of 3 to 100 amino acid residues
25 or more amino acid residues in defined sequence
C530S300000, C530S328000, C530S329000, C530S330000, C530S333000, C514S002600
Reexamination Certificate
active
06326466
ABSTRACT:
FIELD OF THE INVENTION
The invention relates to compositions and methods to inhibit activation of double-stranded RNA dependent protein kinase (PKR) to stimulate cell proliferation under conditions of cell cycle arrest, quiescence, reduced growth or cell death. The invention also relates to methods of protecting cells from HIV-1 pathogenesis using inhibitors of PKR.
BACKGROUND OF THE INVENTION
Double-stranded RNA dependent protein kinase (alternatively, “PKR”) is a serine/threonine protein kinase which exerts antiviral and anticellular functions. It can be induced by interferon (Meurs, et al.,
Cell
62:379-390 (1990); Sen, et al.
J. Biol. Chem
. 267:5017-5020 (1992); Hovanessian, A. G.,
J. Interferon Res
. 9:641-647 (1989)). PKR is involved in regulating a number of physiologic processes. These include cell growth and differentiation (Petryshyn, et al.,
Proc. Natl. Acad. Sci. USA
85:1427-1431 (1988); Petryshyn, et al.,
J. Biol. Chem
. 259:14736-14742 (1984); Judware, et al.,
Mol. Cell. Biol
. 11:3259-3267 (1991), tumor suppression (Koromilas, et al.,
Science
257:1685-1689 (1992); Meurs, et al.,
Proc. Natl. Acad. Sci. USA
90:232-236 (1993)), and modulation of signal transduction pathways (Leonardo, et al.,
Cell
57:287-294 (1989); Kumar, et al.,
Proc. Natl. Acad. Sci. U.S.A
. 91:6288-6292 (1994); Maran, et al.,
Science
265:789-792 (1994)).
These cellular effects of PKR have generally been attributed to translational regulation (Farrell, et al.,
Cell
11:187-200 (1977); Petryshyn, et al.,
Methods Enzymol
. 99:346-362 (1983); Samuel, C. E.,
Proc. Natl. Acad. Sci. U.S.A
. 76:600-604 (1979)). In the presence of low concentrations of double-stranded RNA (dsRNA), divalent cations and ATP, PKR undergoes a phosphorylation which is required to convert the enzyme from a latent to an active protein kinase (Edery, et al.,
Cell
56:303-312 (1989); Lebleu, et al.,
Proc. Natl. Acad. Sci. USA
73:3107-3111 (1976); Petryshyn, et al.,
Methods Enzymol
. 99:346-362 (1983)). Paradoxically, the phosphorylation and activation is prevented by high concentrations of dsRNA (Farrell, et al.,
Cell
11:187-200 (1977); Hunter, et al.,
J. Biol. Chem
. 250:7887-7891 (1975)). Once activated, PKR phosphorylates the alpha subunit of the eukaryotic initiation factor 2 (elF-2 alpha) (Farrell, et al.,
Cell
11:187-200 (1977); Lebleu, et al.,
Proc. Nat. Acad. Sci. USA
73:3107-3111 (1976); Petryshyn, et al.,
Methods Enzymol
. 99:346-362 (1983)), which in turn, results in inhibition of protein synthesis (London, et al. (Boyer, et al. (eds)),
The Enzymes
, vol. 18. Academic Press, New York (1987); Hershey, J. W.,
J. Biol. Chem
. 264:20823-20826 (1989)). The antiviral effect of PKR is believed to be mediated the phosphorylation of elF-2 alpha. However, it is not known whether PKR's anticellular effect is due to phosphorylation of elF-2 alpha, 1 kappa B or another unknown substrate (Lee, et al.
Virology
231:81-88 (1997)).
The mechanism by which PKR interacts with dsRNA is unclear. Neither the spatial, structural or sequence requirements within the RNA or the protein itself are sufficiently resolved to fully understand the dynamics of this interaction. Since a diverse group of viral RNAs interact and modulate the activity of PKR (Clarke, et al.,
Nucleic Acids Res
. 19:243-248 (1991); Kitajewski, et al.,
Cell
45:195-200 (1986); Hovanessian, A. G.,
J. Interferon Res
. 9:641-647 (1989); Hunter, et al.,
J. Biol. Chem
. 250:7887-7891 (1975); SenGupta, et al.
Nucleic Acids Res
. 17:969-978 (1989); Roy, et al.,
J. Virol
65:632-640 (1991); Edery, et al.,
Cell
56:303-312 (1989); Judware, et al.,
J. Interferon Res
. 13:153-160 (1993); Biscboff, et al.,
Virology
172:106-115 (1989)), there does not appear to be sequence specificity. However, there is a dependency on both the length of the double-strandedness and its secondary structure (Manche, et al.,
Mol. Cell. Biol
. 12:5238-5248 (1992); Ghadge, et al.,
J. Virol
. 68:4137-4151 (1994); Hunter, et al.,
J. Biol. Chem
. 250:7887-7891 (1975); Edery, et al.,
Cell
56:303-312 (1989)). Tertiary structure is also likely to be important. Several viral RNAs inhibit the activation of PKR (Kitajewski, et al.,
Cell
45:195-200 (1986); Clarke, et al.,
Nucleic Acids Res
. 19:243-248 (1991); Ghadge, et al.,
J. Virol
. 68:4137-4151 (1994)), while others are efficient activators (Hovanessian, A. G.,
J. Interferon Res
. 9:641-647 (1989)). The TAR sequence of HIV-1 mRNA transcript has been shown to both activate (Edery, et al.,
Cell
56:303-312 (1989); SenGupta, et al.
Nucleic Acids Res
. 17:969-978 (1989); Judware, et al.,
J. Interferon Res
. 13:153-160 (1993)) and prevent activation (Gunnery, et al.,
Proc. Natl. Acad. Sci. USA
87:8687-8691 (1990)) of PKR at low concentrations.
Both human (Meurs, et al.,
Cell
62:379-390 (1990)) and murine PKR (Feng, et al.,
Proc. Natl. Acad. Sci. USA
89: 5447-5451 (1992); Baier, et al.,
Nucleic Acids Res
. 21:4830-4835 (1993)) have been cloned and sequenced and these two cDNAs share extensive nucleotide sequence identity (Feng, et al.,
Proc. Natl. Acad. Sci. USA
89: 5447-5451 (1992)). Results from several studies have reported that the RNA-binding domain of PKR is localized to the N-terminal portion of the kinase. Feng, et al.,
Proc. Natl. Acad. Sci. USA
89: 5447-5451 (1992); McCormack, et al.,
Virology
188:47-56 (1992); Patel, et al.,
J. Biol. Chem
. 269:18593-18598 (1994); Green, et al.,
Genes Dev
. 6:2478-2490 (1992); Patel, et al.,
J. Biol. Chem
. 267:7671-7676 (1992). Although deletions of several short portions of PKR sequence rich in positively charged residues have been shown to diminish dsRNA-induced PRK activation, no discrete PKR region or amino acid sequence motif which is both necessary and sufficient to bind to regulatory dsRNA was known prior to this invention (Feng, et al.,
Proc. Natl. Acad. Sci. USA
89: 5447-5451 (1992)).
Thus, prior to this invention, the existence of a defined linear, non-conformationally dependent dsRNA-binding region of PKR, which is both necessary and sufficient to bind to dsRNA, was unknown.
Furthermore, PKR antagonists were unknown. As PKR is a regulator of cell quiescence and cell death, such antagonists would be valuable for treating diseases or conditions associated with premature or induced cell death, such as the T cell depletion due to HIV-1 infection.
Thus, there exists a great need for inhibitors of PKR. The present invention fulfills these and other needs.
SUMMARY OF THE INVENTION
The invention relates to compositions and methods to inhibit activation of double-stranded RNA dependent protein kinase (PKR) to stimulate cell proliferation under conditions of cell cycle arrest, quiescence, reduced growth or cell death. The invention also relates to methods of protecting cells from HIV-1 pathogenesis using inhibitors of PKR. The invention further relates to methods for inhibiting apoptosis mediated by PKR.
In one aspect, the present invention is directed to an isolated protein kinase double-stranded RNA dependent protein kinase (PKR) peptide antagonist of less than about 50 amino acid residues in length and comprising at least about 8 contiguous amino acid residues from a sequence selected from the group consisting of: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, or a conservatively modified variant thereof, wherein said variant forms a complex with regulatory RNA. In another embodiment of the invention, the peptide of claim
1
includes at least about 9 contiguous residues from the sequence or a conservatively modified variant thereof. In alternative embodiments, the invention includes: the peptide of claim
1
, wherein said peptide comprises the sequences of claim
1
or a conservatively modified variant thereof; and, the peptide of claim
1
, wherein said peptide is the sequence shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, or SEQ ID NO:6.
In an additional embodiment, the invention includes the peptide of claim
1
comprising no more than about 25 amino acids. In another embodim
Bottaro Donald P.
Petryshyn Raymond
G. Anita
Hunt Jennifer
The United States of America as represented by the Secretary of
Townsend and Townsend / and Crew LLP
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