Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Peptide containing doai
Reexamination Certificate
2001-01-25
2004-06-08
Low, Christopher S. F. (Department: 1653)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Peptide containing doai
C514S002600, C530S350000, C530S300000, C536S023100, C435S006120, C435S069100, C435S015000, C435S007100
Reexamination Certificate
active
06747005
ABSTRACT:
The present invention relates to screening methods, peptides, mimetics, and methods of use based on the surprising discovery and characterisation of an interaction between known proteins, and thus numerous cellular processes of interest in therapeutic contexts. The proteins in question are CBP and Importin &agr;, it further being shown herein that CBP acetylates Importin &agr; at residues affecting nuclear localisation of Importin &agr;.
Various enzymes have been identified which acetylate histones, so called histone acetyl-transferases or “HAT”s. There are four families known of enzymes with such activity. These are GCN5 and P/CAF (Brownell, et al (1996),
Cell,
84: 843-831 and Yang, et al (1996),
Nature,
382: 319-3241), CBP and p300 (Bannister and Kouzarides (1996),
Nature,
384: 641-643 and Ogryzko, et al (1996),
Cell,
87: 953-959), SRCl and ACTR (Chen, et al (1997),
Cell,
90: 569-580 and Spencer, et al (1997),
Nature,
389: 194-198) and TAF250 (Mizzen, et al (1996),
Cell,
87: 1261-1270). The precise in vivo targets of these enzymes are largely unknown. In vitro experiments suggest that recombinant enzymes may have specificity for distinct lysines within the same histone. Precisely how acetylation of a particular histone increases transcription is not known.
Recently, evidence has been provided that some proteins other than histones are acetylated. The p53 transcription factor and the basal transcription factors TFIIE and TFIIF have been shown to be acetylated (Imhof, et al (1997),
Current Biology,
7: 689-692). In the case of p53 it has been shown that acetylation increases the DNA binding capacity of the protein (Gu and Roeder (1997),
Cell,
90: 595-606).
The present invention is based on the surprising discovery that CBP interacts with and acetylates a molecule which is not a transcription factor—Importin &agr;—and moreover that the acetylated residues are important for Importin &agr; function.
Experimental work on this is described below and leads to various aspects of the present invention in which there is provided for modulation of interaction between CBP and Importin &agr;, particularly acetylation of Importin &agr; by CBP.
Various aspects of the present invention provide for the use of CBP and Importin &agr; in screening methods and assays for agents which modulate interaction between CBP and Importin &agr;, particularly acetylation of Importin &agr; by CBP, and agents which modulate the ability of Importin &agr; to enter and/or exit the nucleus, and especially ability of Importin &agr; to transport into the nucleus a cargo molecule.
Importin &agr; is a shuttling NLS (“nuclear localisation signal”) receptor which mediates the import into the nucleus of NLS-containing proteins. Exemplary NLS's include the SV40 T antigen NLS (PKKKRKV) (SEQ ID NO:1) and the bipartite nucleoplasmin NLS (KRPAAIKKAGQAKKKK) (SEQ ID NO:2). NLS-containing proteins bind Importin &agr; in the cytoplasm and are imported into the nucleus.
Importin &agr; is also known as Karyopherin &agr;, Kap60, and Srp1. In the cytoplasm, Importin &agr; binds the NLS-containing cargo protein and also Importin &bgr;. Importin &bgr; docks the heterotrimeric complex to the nuclear pores. At the nucleoplasmic side, the complex disassembles, the cargo is released and Importin &agr; returns to the cytoplasm bound to its export factor, CAS, to start a new round for import (for reviews see Görlich, 1998
EMBO J.
17: 2721-2727; Ullmann et al., 1997,
Cell
90: 967-970).
Identification of key residues in Importin &agr; acetylated by CBP may also be used in the design of peptide and non-peptidyl agents which modulate, particularly inhibit, acetylation of Importin &agr; (i.e. the transfer of an acetyl group from acetylcoenzyme A to the &egr;-amino group of a lysine residue in the protein) by CBP or other acetylase enzyme, as discussed further below.
Methods of obtaining agents able to modulate interaction between CBP and Importin &agr; include methods wherein a suitable end-point is used to assess interaction in the presence and absence of a test substance. Assay systems may be used to determine CBP acetylase activity and/or CBP interaction with Importin &agr; and/or acetylation of Importin &agr; by one or more other acetylases. For acetylation assays, full-length Importin &agr;, truncated portions of Importin &agr;, or portions of Importin &agr; fused to other proteins (e.g. GST), or a suitable variant or derivative of any of these may be used. Peptide acetylation assays may be developed using peptides that correspond to the acetylated regions of Importin &agr;. The acetylation of any of the above may be assayed by any of a variety of procedures such as discussed below and may be adapted to high throughput screening approaches. Generally of most interest is modulation of the acetylation of Importin &agr; by CBP or other acetylase. Detailed disclosure in this respect is included below. It is worth noting, however, that combinatorial library technology provides an efficient way of testing a potentially vast number of different substances for ability to modulate an interaction with and/or activity of a polypeptide. Such libraries and their use are known in the art, for all manner of natural products, small molecules and peptides, among others. The use of peptide libraries may be preferred in certain circumstances.
Given the results reported herein on which the present invention is based, activators and inhibitors of CBP-associated acetylase activity or other acetylase able to acetylate Importin &agr; may be identified and appropriate agents may be obtained, designed and used for any of a variety of purposes.
Modulation of Importin &agr; function may be used in principle to influence delivery to the nucleus of any NLS-containing protein. A very large number of nuclear proteins can enter the nucleus from the cytoplasm by means of recognition of the NLS by Importin &agr;. Such proteins include factors involved in replication, DNA repair, recombination, apoptosis, cell proliferation and many other processes.
NLS-containing proteins whose import into the nucleus may be manipulated by modulation of Importin &agr; acetylation in accordance with the present invention include transcription factors which are held in an inactive state in the cytoplasm and are only functional when translocated to the nucleus. Examples of such proteins include NF-KB, NFAT, SMAD, STAT and &bgr;-Catenin (See for example Clevers and Van de Wetering, December 1997, TIG 13(12): 485-489; Heldin et al., 1997,
Nature
390: 465-470; Darnell, 1997,
Science
277: 1630-1635; Ghosh et al., 1998,
Annu. Rev. Immunol.
16: 225-60; Rao et al., 1997,
Annu. Rev. Immunol
15: 707-47. By way of example, &bgr;-Catenin stimulates transcription of an oncogenic pathway when it is nuclear. Mutations found in &bgr;-Catenin stabilise the protein, increase its nuclear import and stimulate its transcriptional activation capacity.
Control of cellular proliferation is of course of interest for treatment of neoplasias, tumours, cancer, psoriasis, arteriosclerosis and other hyper-proliferative disorders. Other disorders that may be treated by modulation of nuclear import in accordance with the present invention include any in which a factor imported into the nucleus plays a role.
Thus, various methods and uses of modulators, which inhibit or potentiate interaction of CBP and Importin &agr;, and modulators that affect acetylation of Importin &agr;, are provided as further aspects of the present invention. The purpose of disruption, interference with or modulation of interaction between CBP and Importin &agr;, particularly the acetylation of Importin &agr; by CBP may be to modulate any activity mediated by virtue of such interaction, as discussed above and further below. Acetylation of Importin &agr; by one or more other acetylases may be modulated for the same or similar purposes.
The full amino acid sequence of the CBP protein has been elucidated and is set out in Chrivia et al. (1993)
Nature
365: 855-859. Residues 1098-1758 of CBP have been shown to include CB
Chroma Therapeutics Limited
Kam Chih-Min
Low Christopher S. F.
Nixon & Vanderhye PC
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