Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Having -c- – wherein x is chalcogen – bonded directly to...
Reexamination Certificate
1996-10-15
2001-10-02
Raymond, Richard L. (Department: 1624)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Having -c-, wherein x is chalcogen, bonded directly to...
C514S245000, C514S261100, C514S274000, C514S634000, C514S635000, C514S646000, C530S345000, C530S402000
Reexamination Certificate
active
06297253
ABSTRACT:
1 FIELD OF THE INVENTION
The field of the present invention concerns compounds that react with specific sequences in proteins. The present invention more particularly concerns a class of compounds that react, under physiologic conditions, with proteins having adjacent or neighboring basic amino acid sequences. The compounds of the invention can be used to label specifically such proteins for research purposes and to disrupt their function for pharmacologic purposes. The compounds of the invention can be used for targeted inactivation of nuclear localization signal in specific proteins or molecular complexes. The compounds of the invention can also be used to treat infectious diseases such as HIV infection and malaria.
2 BACKGROUND TO THE INVENTION
2.1 The Derivatization of Proteins
Those skilled in the art will appreciate that there are many compounds that can react with specific amino acid residues in proteins, e.g., with sulfhydryl, amino, carboxyl moieties. These reagents are substrate specific, in the sense that each reacts only with one or a few specific amino acids wherever they occur within a protein's sequence. However, the reactivity of such reagents is not affected by the adjacent or neighboring amino acids that form the environment of the reactive moiety. Thus, the reactivity of such compounds is not context or neighborhood specific.
2.2 Nuclear Importation
The function of an intracellular protein is usually the result of the overall three dimensional (tertiary) structure of the protein. However, nuclear importation is determined by the simple presence of a short sequence, called a nuclear localization signal (NLS), which functions relatively independently of its position relative to the remainder of the structure of object that is imported. In eukaryotic cells all proteins are made in the cytoplasm, which is outside of the nucleus. In general, those proteins larger than 40 kD that are specifically localized in the nucleus of the cell must be actively imported into the nucleus through the nuclear membrane from the cytoplasm via an ATP-dependent mechanism that is independent of cell division. The proteins, and other objects, that are imported have a nuclear localization signal (NLS), usually located within the NH
2
terminal segment of the protein. Several such sequences are known:
a. PKKKRKV from large T antigen of SV40 and other papillomaviruses such as JC, see Kalderon, D., et al., 1984, Cell 39:499-509;
b. [AV]KRPAATKKAGQAKKKK[LD] from nucleoplasmin, in which only one of the two bracketed sequences is required, Dingwall, C., et al., 1988, J. Cell Biol. 107:841-49;
c. PRRRRSQS from hepatitis B HbcAg- Yeh, C. T., 1990, J. Virol.
d. KRSAEGGNPPKPLKKLR from the retinoblastoma gene product p110
rb1
—Zacksenhaus E. et al., 1993, Mol. Cell. Biol. 13:4588
e. KIRLPRGGKKKYKLK from the matrix protein of HIV-1, Bukrinsky, M. I., et al., 1993, Nature 365:666.
Other viruses that contain NLS sequences include influenza virus (NP, PA, PB1, PB2 proteins which have lysine-rich NLS similar to SV40), hepatitis delta virus (HDAg, which has the sequence PKKKXKK), parvoviruses such as RA1 (NS, VP proteins which have lysine-rich NLS similar to SV40), Herpes simplex and measles virus. The recognition of an NLS sequence is largely independent of the detailed structure of the object which includes it and of its site of attachment. Goldfarb, D. S. et al., 1986, Nature 332:641-44; Lanford, R. E., 1986, Cell 46:575. Mere juxtaposition of the amino acids of the NLS is not sufficient for function, for example NLS function is generally not conferred by the peptide having the same sequence of amino acids in the opposite order as the NLS sequence. Adam, S. A. et al., 1989, Nature 337:276-79.
The primary structure, i.e., the linear sequence, of the NLS most frequently contains consecutive lysines, the N
&egr;
moieties of which presumably closely approach one another, i.e., they are neighbors. However, certain functional NLS peptides lack consecutive lysines. Robbins, J., et al., 1991, Cell 64:615-23. Presumably the secondary and tertiary structure of these so called “bipartite” NLS peptides gives rise to neighboring N
&egr;
moieties, which may be important for their activity.
Docking and subsequent movement of proteins across the nuclear pore complex require transport factors. Import of NLS-containing proteins across the nuclear pore complex is mediated by karyopherin &agr;&bgr; heterodimers (also termed NLS receptor/importin) which bind NLS-containing proteins in the cytosol and target them to the nucleus (Gorlich, D., et al., 1995, Curr. Biol. 5:383-392; Radu, A., et al., 1995, Proc. Natl. Acad. Sci. 92:1765-1773). Karyopherin &agr; binds the NLS (Adam and Gerace, 1991, Cell 66:837-847) whereas karyopherin &bgr; enhances the affinity of &agr; for the NLS (Rexach and Blobel, 1995, Cell 83:683-692) and mediates docking of karyopherin-NLS protein complexes to nucleoporins (a collective term for nuclear pore complex proteins) that contain FXFG peptide repeats. The GTPase Ran and its interacting protein p10 (also termed NTF2) (Moore and Blobel, 1994, Proc. Natl. Acad. Sci. 91:10212-10216) impart mobility to the translocation process by catalyzing the disruption of karyopherin &agr;&bgr; heterodimers that have docked to a nucleoporin (Nerhbass and Blobel, 1996, Science 272:120-122). Partial reactions of the nuclear import can be reproduced in vitro using solution binding assays and recombinant karyopherins (Rexach and Blobel, 1995, supra).
Two inhibitors of the nuclear localization process have been described. Nuclear localization has been inhibited by lectins (e.g., wheat germ agglutinin (WGA)) that bind to the O-linked glycoproteins associated with nuclear localization. Dabauvalle, M.-C., 1988, Exp.Cell Res. 174:291-96; Sterne-Marr R., et al., 1992, J.Cell Biol. 116:271. The nuclear localization process, which also depends upon the hydrolysis of GTP, is blocked by a non-hydrolyzable analog of GTP, e.g., (&ggr;-S)GTP, Melchior, F., 1993, J.Cell Biol. 123:1649.
However, neither (&ggr;-S)GTP nor WGA can be used as pharmaceuticals. Proteins, such as WGA, can be introduced into the interior of a cell only with considerable difficulty. The same limitation applies to thiotriphospates such as [&ggr;-S]GTP. Further, GTPases are involved in a multitude of cell processes and intercellular signaling, thus, the use of a general inhibitor of GTPases would likely lead to unacceptable side effects.
2.3 The Significance of Nuclear Importation in HIV-1 Infections
Although HIV-1 is a retrovirus, it and other lentiviruses must be distinguished from viruses of the onco-retrovirus group, which are not associated with progressive fatal infection. For example, lentiviruses replicate in non-proliferating cells, e.g., terminally differentiated macrophages, Weinberg, J. B., 1991, J.Exp. Med. 172:1477-82, while onco-retroviruses, do not. Humphries, E. H., & Temin, H. M., 1974, J.Virol. 14:531-46. Secondly, lentiviruses are able to maintain themselves in a non-integrated, extrachromosomal form in resting T-cells. Stevenson, M., et al., 1990, EMBO J. 9:1551-60; Bukrinsky, M. I., et al., 1991, Science 254:423; Zack, J. L., et al., 1992, J.Virol. 66:1717-25. However, it is unclear whether this phenomenon is related to the presence of latently infected peripheral blood lymphocytes (PBL) in HIV-1 infected subjects, wherein the virus is present in a provirus form. Schnittman, S. M., 1989, Science 245:305; Brinchmann, J. E., et al., 1991, J.Virol. 65:2019; Chapel, A., et al., 1992 J. Virol. 66:3966.
The productive infection of a cell by a retroviruses involves the steps of penetration into the cell, synthesis of a DNA genome from the RNA genetic material in the virion and insertion of the DNA genome into a chromosome of the host, thereby forming a provirus. Both lenti- and oncoretroviruses gain access to the host cell's nucleus during mitosis when the nuclear membrane dissolves. However, the lentiviruses are also able to cross the nuclear membrane because viral proteins containing nuclear local
Bukrinsky Michael L.
Cerami Anthony
Ulrich Peter
Kelber Steven B.
Piper Marbury Rudnick & Wolfe LLP
Raymond Richard L.
The Picower Institute for Medical Research
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