Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Peptide containing doai
Reexamination Certificate
1998-08-12
2001-07-10
Russel, Jeffrey E. (Department: 1653)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Peptide containing doai
C435S015000, C436S086000, C514S012200, C514S013800, C514S014800, C514S015800, C530S300000, C530S324000, C530S328000
Reexamination Certificate
active
06258776
ABSTRACT:
The present invention relates to kinase proteins of the nuclear Dbf2-related (Ndr) protein kinase family and to the activation thereof in response to calcium signalling.
Reversible protein phosphorylation is a major mechanism for the co-ordinated control of many fundamental cellular functions in eukaryotic organisms, including metabolism, growth, and differentiation. The phosphorylation status, and consequently the activity, of specific target proteins is regulated by the opposing actions of protein kinases and protein phosphatases. Generally, these enzymes are specific either for serine/threonine or for tyrosine phosphoacceptors, although some dual specificity kinases and phosphatases have also been described. The importance of phosphorylation cascades is reflected by the finding that many kinases, phosphatases, and the signal transduction pathways in which they participate have been highly conserved during the course of evolution. In recent years, interest has focused on the role of protein phosphorylation in the control of the cell cycle; a number of cellular proto-oncogenes encode members of the serine/threonine kinase family and it has become increasingly clear that certain serine/threonine kinases function as key components of the cell cycle regulatory network. Therefore, the complete delineation of these pathways is an important aim for the understanding of oncogenesis and tumour progression.
Ndr is a nuclear serine/threonine protein kinase that was recently cloned from human brain,
D. melanogaster
and
C. elegans
cDNA libraries (Millward, T. A., et aL, (1995)
Proc. Natl. Acad. Sci. USA
92, 5022-5026). Although the high level of conservation of Ndr between these species implies an important role in cellular physiology, its function is currently unknown. Possible clues are, however, suggested by its phylogenetic relationship to several serine/threonine kinases from lower eukaryotes which have been identified in genetic screens as essential for normal cell growth and morphology. This emerging subfamily of protein kinases presently comprises, in addition to Ndr, Wts/lats (a tumour suppressor gene product from
D. melanogaster
), cot-1 (required for growth by hyphal elongation in N. crassa) and Dbf2/Dbf20 (budding yeast cell cycle-regulated kinases with a metaphase/anaphase function) (Justice, R. W., et al., (1995)
Genes Dev.
9, 534-546; Xu, T., et al., (1995)
Development
121, 1053-1063; Yarden, O., et al., (1992)
EMBO J,
11, 2159-2166; and Toyn, J. H. and Johnston, L. H. (1994)
EMBO J.
13,1103-1113). These kinases share 40-60% amino acid identity in their catalytic domains and show several conserved features outside of their catalytic domains. For example, a hallmark of this group of kinases is that each contains an “insert” in its catalytic domain, located between subdomains VII and VIII, which is not present in other protein kinases. Two mammalian kinases, ROK&agr; (Leung, T., et al., (1995)
J. Biol. Chem.
270, 29051-29054) and the myotonic dystrophy kinase DMK (Brook, J. D., et al., (1992) Cell 68, 799-808), are also related to this subfamily, although they do not contain the kinase domain insert. In the superfamily of serine/threonine protein kinases, this kinase subfamily falls within the second messenger-regulated (“AGC”) group (Hanks, S. K. and Hunter, T. (1995) in
The protein kinase facts book: protein
-
serine kinases
(Hardie, G. and Hanks, S. K., eds) pp. 7-47, Academic Press Ltd., London). However, while genetic studies have been instrumental in defining the possible functions of these kinases, their regulation is only partially understood. ROK&agr; is activated by interaction with the GTP-bound form of RhoA; however, binding to RhoA is mediated by a domain of ROK&agr; not present in the other kinases (Leung et al.,
Op. Cit
.), suggesting that these kinases are regulated by other mechanisms.
Binding of growth factors and peptide hormones to cell surface receptors leads to transient increases in the intracellular concentrations of several “second messengers.” One such second messenger is Ca
2+
, which elicits multiple cellular responses primarily by binding to a large family of EF hand-containing proteins (Hunziker, W. and Heizmann, C. W. (1991)
Trends Biochem. Sci.
16, 98-103; James, P., et al., (1995)
Trends Biochem. Sci.
20, 38-42). This family includes, among others, calmodulin (CaM) and S100 proteins. Binding of Ca
2+
to these proteins induces a conformational change in the polypeptide which exposes a hydrophobic surface capable of binding to secondary effector proteins. Ca
2+
fluxes play an important role in the regulation of cell motility, cell division, transcription, protein synthesis and apoptosis. This diversity of function may be possible because Ca
2+
binds to multiple classes of EF hand proteins, each of which can, in turn, regulate multiple downstream effectors.
WO96/19579 discloses that Ndr protein kinase is capable of binding to calmodulin, a major calcium-binding protein of the EF hand family. This suggested that calmodulin may play a role in the activation of Ndr. However, this determination was made without access to an efficient assay for Ndr activity, because none existed at the time; the substrates commonly used for other protein kinases, such as histone, myelin basic protein and casein, are not substrates for Ndr. Moreover, the site of calmodulin binding to Ndr was not disclosed.
SUMMARY OF THE INVENTION
Employing a novel and effective test for Ndr activity, it has now been determined that calmodulin is a weak, but not a major, in vitro activator of Ndr. Ndr is, however, regulated by Ca
2
+concentration, and is activated by two calmodulin-related polypeptides, S100B and S100 in a calcium-dependent manner. Both S100B and S100 bind Ndr in the N-terminal region, exerting their activatory activity at this site. The binding site appears to coincide or overlap with the calmodulin binding site, suggesting that both calmodulin and the S100 proteins bind Ndr in a similar manner. Furthermore, it has now been shown that Ndr is negatively regulated by protein phosphatase 2A (PP2A), a phosphoserine/phosphothreonine phosphatase.
In a first aspect of the present invention, therefore, there is provided a method of modulating the activity of a polypeptide comprising the activating domain of an Ndr family kinase, by influencing the binding of an EF hand-containing calcium binding protein to the Ndr family kinase activating domain.
In a further aspect, the invention provides agents capable of influencing the binding of EF-hand containing polypeptides to the N-terminal binding domain of an Ndr family kinase. Preferably, the agents may be based on peptides derived from the binding domain.
Moreover, the invention provides a method for screening potential activators or repressors of Ndr family kinase activity, by (a) screening compounds for the ability to bind to the N-terminal binding domain of Ndr family kinase, or to modulate the binding of EF hand-containing polypeptides to this domain, and/or by (b) screening candidate compounds for the ability to modulate the activity of Ndr family kinase in vitro.
Ndr activity may be measured according to a further aspect of the invention, which provides a method for assessing Ndr activity comprising determining the ability of Ndr to phosphorylate a target peptide B
1
B
2
B
3
x B
4
B
5
x S n x, where at least two of B
1-5
are basic amino acids, the remainder of B
1-5
being any amino acid, x is any amino acid, n is any amino acid except proline and S is serine. Also provided are peptides suitable for use in this aspect of the invention, having the above general formula.
Moreover, the present invention provides a pharmaceutical composition comprising an EF-hand containing calcium binding protein, or a molecular mimic or regulator thereof, as well as the use of an EF-hand containing calcium binding protein, or a molecular mimic or regulator thereof in the manufacture of a composition for modulating the activity of a polypeptide comprising the activating domain of an Nd
Hemmings Brian Arthur
Millward Thomas Anders
Novartis AG
Pfeiffer Hesna J.
Russel Jeffrey E.
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