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US 2004/0176301 A1Published application
p21 peptides
AssigneeCyclacel Limited
InventorsDaniella I. Zheleva, Peter M. Fischer, Campbell McInnes, Martin J.I. Andrews, Weng C. Chan, Gail E. Atkinson
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Abstract
The present invention relates to p21 derived peptides capable of inhibiting CDK/cyclin complexes, particularly cyclins A or E/CDK2, by modifying the interaction with their substrates. The peptides are derived from a C-terminal region of p21 and display selectivity for cyclin/CDK2 inhibition over cyclin/CDK4 inhibition. Variants of such peptides particularly involving certain alanine replacements are shown to be particularly potent.
Claims54
A peptide of formula I
- N1DFYHSKRRLIFN2 (formula I) (SEQ ID No. 4),
- comprising the motif XLXF,
- wherein N1 and N2 are independently a natural or non-natural amino acid or nothing;
- or the peptide of formula I having up to 8 amino acid residues deleted from the N-terminal end;
- and variants thereof wherein at least one amino acid residue is replaced by an alternative natural or non-natural replacement amino acid residue, with the proviso that the motif XLXF is retained, wherein X refers to any natural or unnatural amino acid.
2 more elements
A peptide of formula
- DFYHSKRRLIF (SEQ ID No. 1),
- comprising the motif XLXF,
- or such a peptide
- (i) bearing a further amino acid residue at either end;
- and,
- (ii) having up to 7 amino acid residues deleted from the N-terminal end;
- and variants thereof wherein at least one amino acid residue is replaced by an alternative natural or unnatural replacement amino acid residue, with the proviso that the motif XLXF is retained, wherein the peptide of SEQ ID No. 1 is modified by at least one of;
- deletion, addition or substitution of one or more amino acid residues, or by substitution of one or more natural amino acid residues by the corresponding D-stereomer or by a non-natural amino acid residue, chemical derivatives of the peptides, cyclic peptides derived from the peptides or from the peptide derivatives, dual peptides, multimers of the peptides and any of said peptides in the D-stereomer form, or the order of the final two residues at the C-terminal end are reversed.
5 more elements
A peptide of formula II;
- X1X2X3RX4LX5F (formula II) (SEQ ID No. 2)
- wherein X1, X3, X4 and X5 may be any amino acid and X2 is serine or alanine;
- and variants thereof.
A peptide of formula;
- X1X2X3RX4LX5F (SEQ ID No. 2)
- wherein X1, X3, X4 and X5 may be any amino acid and X2 is serine or alanine;
- and variants thereof, wherein the peptide is modified by at least one of a deletion, addition or substitution of one or more amino acid residues, or by substitution of one or more natural amino acid residues by the corresponding D-stereomer or by a non-natural amino acid residue, chemical derivatives of the peptides, cyclic peptides derived from the peptides or from the peptide derivatives, dual peptides, multimers of the peptides and any of said peptides in the D-stereomer form, or the order of the final two residues at the C-terminal end are reversed.
A peptide of formula;
- X1X2X3RX4LX5F (SEQ ID No. 2)
- wherein X1, X3, X4 and X5 may be any amino acid and X2 is serine or alanine;
- and variants thereof, wherein:
- (a) X1 is deleted or is any amino acid,
- (b) X2 is serine or alanine or a straight or branched chain amino acid,
- (c) X3 is a basic amino acid or straight chain aliphatic amino acid,
- (d) R is unchanged or conservatively substituted (by basic amino acids),
- (e) X4 is any amino acid that is capable of providing at least one site for participating in hydrogen bonding,
- (f) L is unchanged or conservatively substituted,
- (g) X5 is any amino acid, or
- (h) F is unchanged or substituted by any aromatic amino acid.
8 more elements
A peptide of formula;
- X1X2X3RX4LX5F (SEQ ID No. 2),
- wherein
- (a) X1 is histidine, deleted or replaced by a natural or unnatural amino acid residue such as alanine, 3-pyridylalanine (Pya), 2-thienylalanine (Thi), homoserine (Hse), phenylalanine, or diaminobutyric acid (Dab),
- (b) X2 is alanine or an alternative natural or unnatural amino acid residue having a smaller or slightly larger aromatic or aliphatic side chain, such as glycine, aminobutyric acid (Abu), norvaline (Nva), t-butylglycine(Bug), valine, isoleucine, phenylglycine (Phg) or phenylalanine,
- (c) X3 is lysine or either a basic residue such as arginine or an uncharged natural or unnatural amino acid residue, such as norleucine (Nle), aminobutyric acid (Abu) or leucine,
- (d) arginine is replaced by either a basic residue such as lysine or an uncharged natural or unnatural amino acid residue, such as citrulline (Cit), homoserine, histidine, norleucine (Nle) or glutamine,
- (e) X4 is arginine or a natural or unnatural amino acid residue, such as asparagine, proline, serine, aminoisobutyric acid (Aib) or sarcosine (Sar), or an amino acid residue capable of forming a cyclic linkage such as lysine or ornithine,
- (f) leucine is replaced with a natural or unnatural amino acid residue having a slightly larger aromatic or aliphatic side chain, such as norleucine, norvaline, cyclohexylalanine (Cha), phenylalanine or 1-naphthylalanine (1Nal),
- (g) X5 is isoleucine or an alternative natural or unnatural amino acid residue having a slightly larger aromatic or aliphatic side chain, such as norleucine, norvaline, cyclohexylalanine (Cha), phenylalanine or 1-naphthylalanine (1Nal),
- (h) phenylalanine is replaced with a natural or unnatural amino acid such as leucine, cyclohexylalanine (Cha), homophenylalanine (Hof), tyrosine, para-fluorophenylalanine (pFPhe), meta-fluorophenylalanine (mFPhe), trptophan, 1-naphthylalanine (1Nal), 2-naphthylalanine (2Nal), biphenylalanine (Bip) or (Tic),
- (i) X5 and the terminal phenylalanine residue are reversed, or
- (j) the peptide is in cyclic form by the formation of a linkage between the side chain of X4 and the C-terminus residue.
9 more elements
A peptide of the formula III or IV;
- H′X2K′R1R2L′X5F (formula III) (SEQ ID No. ______) or H′X2K′R1R2L′FX5 (formula IV) (SEQ ID No. ______)
- or a variant thereof, wherein
- H′ is nothing, His, D-His, Ala, Thi, Hse, Phe, or Dab;
- X2 is Ala, Ser, Abu, Val;
- K′ is Lys, Arg, or Abu;
- R1 is Arg, Lys, or Gln;
- and
- R2 is Arg, forms a cyclic peptide with the C-terminal residue, Ser, or Cit;
- L′ is Leu or Ile;
- X5 is Ile, Leu, Gly, or Ala;
- and
- F′ is Phe, para-fluoroPhe, meta-fluoroPhe, L-Psa, 2-Nap,Dhp, or D-Psa.
9 more elements
An assay for the identification of compounds that interact with a cyclin or a cyclin when complexed with the physiologically relevant CDK, comprising;
- (a) incubating a candidate compound and peptide of formula I;
- X1X2X3RX4LX5F (formula II) (SEQ ID No. 2)
- wherein X1, X3, X4 and X5 may be any amino acid and X2 is serine or alanine;
- and variants thereof or a peptide of the formula III or IV:
- H′X′2K′R1R2L′X′5F′ (formula III) (SEQ ID No. ______) or H′X′2K′R1R2L° F′X′5 (formula IV) (SEQ ID No. ______) or a variant thereof, wherein
- H′ is His, nothing, D-His, Ala, Thi, Hse, Phe, or Dab;
- X′2 is Ala, Ser, Abu, Val;
- K′ is Lys, Arg, or Abu;
- R1 is Arg, Lys, or Gln;
- and
- R2 is Arg, forms a cyclic peptide with the C-terminal residue, Ser, or Cit;
- L′ is Leu or Ile;
- X′5 is Ile, Leu, Gly, or Ala;
- F′ is Phe, para-fluoroPhe, meta-fluoroPhe, L-Psa, 2-Nap,Dhp, or D-Psa.
- and a cyclin or cyclin/CDK complex;
- (b) detecting binding of either the candidate compound or the peptide of formula II or III with cyclin.
13 more elements
An assay for candidate compounds that interact with a cyclin by virtue of forming associations with at least two of the amino acids corresponding to the cyclin A amino acids L253, I206 and R211.
A method of using a cyclin in a drug screening assay comprising:
- (a) selecting a candidate compound by performing rational drug design with a three-dimensional model of said cyclin, wherein said selecting is performed in conjunction with computer modeling;
- (b) contacting the candidate compound with the cyclin;
- and
- (c) detecting the binding affinity of the candidate compound for the cyclin groove;
- wherein a potential drug is selected on the basis of its having a greater affinity for the cyclin groove than that of a peptide of formula II;
- X1X2X3RX4LX5F (formula II) (SEQ ID No. 2)
- wherein X1, X3, X4 and X5 may be any amino acid and X2 is serine or alanine;
- and variants thereof or a peptide of formula III or IV:
- H′X′2K′R1R2L′X′5F′ (formula III) (SEQ ID No. ______) or H′X′2K′R1R2L° F′X′5 (formula IV) (SEQ ID No. ______) or a variant thereof, wherein
- H′ is His, nothing, D-His, Ala, Thi, Hse, Phe, or Dab;
- X′2 is Ala, Ser, Abu, Val;
- K′ is Lys, Arg, or Abu;
- R1 is Arg, Lys, or Gln;
- and
- R2 is Arg, forms a cyclic peptide with the C-terminal residue, Ser, or Cit;
- L′ is Leu or Ile;
- X′5 is Ile, Leu, Gly, or Ala;
- F′ is Phe, para-fluoroPhe, meta-fluoroPhe, L-Psa, 2-Nap,Dhp, or D-Psa.
15 more elements
A method of using a cyclin in a drug screening assay comprising:
- (a) selecting a candidate compound by performing rational drug design with a three-dimensional model of said cyclin, wherein said selecting is performed in conjunction with computer modeling;
- (b) contacting the candidate compound with the cyclin;
- and
- (c) detecting whether said the candidate compound forms associations with at least the amino acids corresponding to the cyclin A amino acids L253, 1206 and R211.
1 more element
An assay for identifying candidate substances capable of inhibiting CDK in a cell, comprising;
- (a) contacting a cell comprising a cyclin or portion thereof or cyclin groove, and a CDK or portion thereof, with a candidate substance under conditions where, in the absence of the candidate substance, the cyclin or portion thereof or cyclin groove and CDK or portion thereof would interact, and
- (b) monitoring any change in the activity of the CDK or portion thereof, wherein inhibition of CDK activity is indicated by one or more of:
- G0 and/o G1/S cell cycle arrest;
- cell cycle-related apoptosis;
- suppression of E2F transcription factor activity;
- hypophosphorylation of cellular pRb;
- and in vitro anti-proliferative effects.
4 more elements
Dependencies are inferred from the claim text.