Cdk5—specific inhibitory peptides

Details Cdk5—specific inhibitory peptides Cdk5—specific inhibitory peptides

1999-04-12

2004-02-17

Brumback, Brenda (Department: 1654)

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

C530S326000, C530S350000, C514S002600, C514S012200, C514S013800

Reexamination Certificate

active

06693166

ABSTRACT:

BACKGROUND OF THE INVENTION
1) Field of Invention
The present invention relates to cyclin-dependent kinase (Cdk) inhibiting peptides that have specific inhibition effect on certain cyclin-dependent kinases. Cdks are key regulatory enzymes in the eukaryotic cell cycle.
2) Description of Prior Art
Cyclin-dependent kinases (Cdks) are key regulatory enzymes in the eukaryotic cell cycle. The activation of a Cdk depends on its association with its specific cyclin partner. The activity of these enzymes is further regulated by an intricate system of protein-protein interactions and phosphorylation (Morgan, D. O., 1995). Members of the Cdk family are closely related by sharing a high level of amino acid sequence identity (40%-70%). In contrast, cyclins are a family of molecules of diverse molecular mass and low sequence identity. Sequence alignments have shown that cyclins share a conserved region of approximately 100 amino acids in the center of the molecule, and this region is called the cyclin box. Recent crystal structures of cyclin A and cyclin H have shown that the cyclin box sequence forms a compact five-helix domain called the cyclin fold. The C-terminal domain of cyclin A also forms the cyclin fold, although there is virtually no sequence similarity between the two cyclin domains. Theoretical predictions have suggested that, like cyclin A, other members of the cyclins also contain two cyclin folds.
Unlike other Cdks, Cdk5 activity has been observed only in neuronal and developing muscle cells although the catalytic subunit of the enzyme is present in many mammalian tissues and cell extracts. Recent experimental evidence has demonstrated that Cdk5 plays important roles in neurite outgrowth, patterning of cortex and cerebellum, and cytoskeletal dynamics. Loss of regulation of Cdk5 has been suggested to be involved in Alzheimer's disease (Lew, J and Wang. J. H., 1995). Active Cdk5 was first purified from brain extract as a heterodimer with subunit molecular masses of 33- and 25-kDa. The 33-kDa subunit was later identified as Cdk5, and the 25-kDa activator (Neuronal Cdk5 activator, Nck5a) was a novel protein with no sequence similarity to any other proteins. The 25-kDa subunit was later found to be proteolytic product of a larger 35-kDa protein. An isoform of Nck5a (Nck5ai) with 57% sequence identity to Nck5a has also been identified. Despite their functional similarity (i.e.: binding and activation of a Cdk), the Cdk5 activators share little sequence similarity to cyclins. Moreover, activation of Cdk5 is independent of phosphorylation of Cdk5 at Ser195 by Cdk activating kinase, CAK. Recently, the activation domain of Nck5a was precisely mapped to the amino acid residues from Glu149 to Asn291. Extensive truncation and site-directed mutation studies of Nck5a, together with computer modelling, strongly suggested that the 142-residue activation domain of Nck5a adopt a cyclin fold structure.
All currently known peptides that have regulatory effects on Cdk5 and Cdk2 are usually ATP/ADP-based. The disadvantages of ATP/ADP-based peptides include their low efficiency of their binding. In addition, they bind to Cdks with low specificity.
OBJECTION OF THE INVENTION
It is an object of the present invention to provide potent Cdk5/Cdk2 specific inhibitory peptides. In particular, it is the object of the present invention to identify the critical binding characteristics of the peptide fragments of Nck5a.
SUMMARY OF INVENTION
Cyclin-dependent Kinases (Cdks) are key regulatory enzymes in the eukaryotic cell cycles. The activation of a Cdk depends on its associating with its specific cyclin partner. In the case of Cdk5, its activation has been shown to occur upon the binding of Nck5a (neuronal Cdk5 activator). A 28-residue peptide encompassing amino acid residues Ala 146 to Asp 173 of Nck5a was discovered to be able to bind to Cdk5 and hence result in kinase inhibition. Additionally, it is found that this peptide could inhibit Cdk2 with an even higher potency than its inhibition of Cdk5. This Nck5a-derived peptide was able to inhibit Cdk5 both during and after reconstitution of the enzyme with Nck5a, suggesting that the peptide does not directly compete with Nck5a for Cdk5. The addition of a 1000-fold excess of the synthetic peptide does not lead to the dissociation of Nck5a from Cdk5 or cyclin A from Cdk2. To identify the molecular basis of kinase inhibition by the peptide, the structure of the peptide in solution was determined by the methods of circular dichrosim and two-dimensional
1
H NMR spectroscopy. The peptide segment adopts an amphiphilic alpha-helical structure from residues Ser149 to Arg162. Four Leu residues and one Phe residue clustered on the hydrophobic face of the helix, and this hydrophobic phase is likely to be the contact area when the peptide binds to Cdk5 and Cdk2. Additionally, a number of peptide analogs were generated, and their inhibitory capacities were also measured. The peptides discovered in this work serve as bio-medical reagents as well as leads for novel drug discovery.
The present invention relates to the identification and characterisation of a 29-residue Cdk inhibitory peptide, which is derived from an internal fragment of Nck5a. This peptide is able to bind to and hence inhibit the Cdk5/Nck5a and Cdk2/cyclinA complexes in a non-competitive manner. The solution structure of the peptide determined by two-dimensional NMR spectroscopy showed that a large part of the peptide adopts an amphipathic alpha-helical structure, and that this helix is likely to be the main contacting area of the peptide that interacts with the enzyme complex.
This Nck5a-derived peptide is the first reported inhibitory peptide of Cdk5. As a result, of the peptides inhibitory specificities against Cdk5 and Cdk2 this peptide possess important and valuable implications as a scientific tool to support biomedical research in cell cycle regulation. These peptides may also be further developed into potential drugs targeting Cdk5/Cdk2 inactivation. Furthermore, the Nck5a-derived peptide possess immediate utility as bio-medical research tools. This peptide can also serve as leads for the development of novel drugs targeting the inhibition of both Cdk5 and Cdk2.


REFERENCES:
Ngo et al., ‘Computational Complexity, Potein Structure Prediction, and the Levinthal Paradox,’ The Protein Folding Problem and Tertiary Structuer Prediction. Ed. K. Merz and L. Le Grand. BirkHauser, Boston MA, pp. 491-495, 1994.*
Rudinger, J. (1976). Peptide Hormones (ed. J.A. Parsons). University Park Press. Baltimore. pp 1-7, 1976.*
Tang et al. ‘Cyclin-dependent kinase 5 (Cdk5) activation domain of neuronal Cdk5 activator. Evidence of the existence of cyclin fold in neuronal Cdk5a activator’, J. Biol. Chem. (1997), 272(19), 12318-12327, May 1997.*
Tsai et al. ‘P35 is a neural-specific regulatory subunit of cyclin-dependent kinase 5,’ Nature (London) (1994), 371(6496), 419-23.*
Patel et al. ‘Pharmacotherapy of Cognative Impairment in Alzheimer's Disease: A Review’, J. Geriatr. Psychiatry Neurol. vol. 8, 1995, pp. 81-92

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