REGULATORS OF CELL DIVISION

Abstract

The present invention relates to peptides and peptidomimetics as well as their medical use in the treatment of hyperproliferative diseases and inflammatory diseases.

Claims

1. A peptide or peptidomimetic comprising the following sequence; (a) X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13-X14 wherein a. X1 is an alpha-amino acid, preferably a proteinogenic amino acid, or preferably Glu or Nmglu; b. X2 is selected from Trp, 2-NaI and 1-NaI; c. X3 is an alpha-amino acid, preferably a proteinogenic amino acid, or preferably selected from Trp, 2-NaI, 1-NaI and Pra; d. X4 is selected from Lys, Cys, D-Cys, Asp, Glu, Dap and (R)-2-(7′-octenyl)Ala; e. X5 is Ile or Lys; f. X6 is an alpha-amino acid, preferably a proteinogenic amino acid, more preferably Leu or Lys; g. X7 is an alpha-amino acid, preferably a proteinogenic amino acid, more preferably Asp or Glu; h. X8 is Ile or Leu; i. X9 is Gln; j. X10 is an alpha-amino acid, preferably a proteinogenic amino acid, more preferably Arg; k. X11 is Asp, Glu, Cys, D-Cys or (S)-2-(4′-pentenyl)Ala; l. X12 is Arg; m. X13 is an alpha-amino acid, preferably a proteinogenic amino acid, more preferably Arg; n. X14 is an alpha-amino acid, preferably a proteinogenic amino acid, more preferably Ala; wherein X2 to X12 of said peptide or peptidomimetic assume an alpha-helix structure under physiological conditions; and wherein a side chain of X4 is covalently connected to a side chain of X11; (b) X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12 wherein a. X1 is selected from Arg, Phe(2-guanidino) and Phe(3-guanidino); b. X2 is selected from Cys, L-propargylglycine (Pra), L-homopropargylglycine (Hpg), allylglycine (Agl), prenylglycine (Pre), crotylglycine (Crt) and (S)-2-amino-4-bromobutyric acid; c. X3 is selected from Ser, His and Dab; d. X4 is an alpha-amino acid, preferably a proteinogenic amino acid, or preferably selected from Trp; e. X5 is an alpha-amino acid, preferably a proteinogenic amino acid, or preferably Gly or D-Ala; f. X6 is Pro or D-Pro; g. X7 is selected from Glu, Arg, Trp, Cit, Orn and pSer; h. X8 is Thr; i. X9 is an alpha-amino acid, preferably a proteinogenic amino acid, or preferably selected from Phe; j. X10 is selected from Trp, 2-NaI and 1-NaI; k. X11 is selected from Cys, (S)-2-amino-4-bromobutyric acid, Dap(N3), 2Abu(γ-N3), Nva(δ-N3), Agl and Crt; l. X12 is selected from Arg, Phe(3-guanidino), Phe(4-guanidino), Cit and Orn; wherein said peptide or peptidomimetic assumes a beta-hairpin structure under physiological conditions, wherein preferably X1 to X4 and X9 to X12 are in beta-sheet conformation and/or a turn is formed by residues X5 to X8; and wherein a side chain of X2 is covalently connected to a side chain of X11; (c) X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13-X14-X15-X16 wherein a. X1 is an alpha-amino acid, preferably a proteinogenic amino acid, more preferably Thr; b. X2 is selected from Ser, His and hSer; c. X3 is selected from Cys, Pra, Hpg, Agl, Crt and (S)-2-amino-4-bromobutyric acid; d. X4 is an alpha-amino acid, preferably a proteinogenic amino acid, more preferably Asp; e. X5 is an alpha-amino acid, preferably a proteinogenic amino acid, more preferably Asp; f. X6 is Pro; g. X7 is Glu, Trp or Aad; h. X8 is Thr; i. X9 is selected from Trp, 2-NaI and 1-NaI; j. X10 is a group consisting of any natural amino acid, preferably a proteinogenic amino acid, more preferably Arg; k. X11 is selected from Trp, 2-NaI and 1-NaI; l. X12 is Asn; m. X13 is selected from Cys, (S)-2-amino-4-bromobutyric acid, Dap(N3), 2Abu(γ-N3), Nva(δ-N3), Agl and Crt; n. X14 is a group consisting of any natural amino acid, preferably a proteinogenic amino acid, more preferably Gln; o. X15 is selected from Arg, Phe(3-guanidino), Phe(4-guanidino) and Cit; p. X16 is an alpha-amino acid, preferably a proteinogenic amino acid, more preferably Val; wherein a side chain of X3 is covalently connected to a side chain of X13; and wherein said peptide or peptidomimetic assumes a helix-turn-helix structure under physiological conditions, wherein preferably X1 to X4 and X10 to X15 are in alpha-helical conformation and/or a turn is formed by X5 to X9; (d) X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13-X14-X25-X16-X17 wherein a. X1 is an alpha-amino acid, preferably a proteinogenic amino acid, more preferably Gly; b. X2 is an alpha-amino acid, preferably a proteinogenic amino acid, more preferably Asp; c. X3 is Thr or hSer; d. X4 is selected from Cys, Pra, Hpg, Agl, Crt and (S)-2-amino-4-bromobutyric acid; e. X5 is an alpha-amino acid, preferably a proteinogenic amino acid, more preferably Asp; f. X6 is an alpha-amino amino acid, preferably a proteinogenic amino acid, more preferably Asp; g. X7 is an alpha-amino amino acid, preferably a proteinogenic amino acid, more preferably Pro; h. X8 is Glu, Trp or Aad; i. X9 is Thr; j. X10 is selected from Trp, 2-NaI and 1-NaI; k. X11 is an alpha-amino acid, preferably a proteinogenic amino acid, more preferably Arg; l. X12 is selected from Trp, 2-NaI and 1-NaI; m. X13 is Asn; n. X14 is selected from Cys, (S)-2-amino-4-bromobutyric acid, Dap(N3), 2Abu(γ-N3), Nva(δ-N3), Pra, Agl and Crt; o. X15 is an alpha-amino acid, preferably a proteinogenic amino acid, more preferably Gln; p. X16 is Arg or Cit; q. X17 is an alpha-amino acid, preferably a proteinogenic amino acid, more preferably His; wherein a side chain of X4 is covalently connected to a side chain of X14; and wherein said peptide or peptidomimetic assumes a helix-turn-helix structure under physiological conditions, wherein preferably X2 to X5 and X11 to X16 are in alpha-helical conformation and/or a turn is formed by X6 to X10; (e) X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13-X14 wherein a. X1 is selected from Cys, Pra, Hpg, Agl, Crt and (S)-2-amino-4-bromobutyric acid; b. X2 is Glu; c. X3 is an alpha-amino acid, preferably a proteinogenic amino acid, or preferably Ala or Aib; d. X4 is an alpha-amino acid, preferably a proteinogenic amino acid, more preferably Gly; e. X5 is Pro; f. X6 is an alpha-amino acid, preferably a proteinogenic amino acid, more preferably Ser; g. X7 is an alpha-amino acid, preferably a proteinogenic amino acid, more preferably Gln; h. X8 is selected from Trp, 2-NaI and 1-NaI; i. X9 is selected from His, Asn and Gln; j. X10 is selected from Cys, (S)-2-amino-4-bromobutyric acid, Dap(N3), 2Abu(γ-N3), Nva(δ-N3), Agl and Crt; k. X11 is an alpha-amino acid, preferably a proteinogenic amino acid, more preferably Thr or Glu; l. X12 is selected from Trp, 2-NaI and 1-NaI; m. X13 is Arg or Cit; n. X14 is an alpha-amino acid, preferably a proteinogenic amino acid, more preferably Tyr; wherein a side chain of X1 is covalently connected to a side chain of X10; wherein said peptide or peptidomimetic assumes a helix-turn structure under physiological conditions, wherein preferably X7 to X14 are in alpha-helical conformation and/or a turn is formed by X1 to X6; (f) X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13-X14 wherein a. X1 is an alpha-amino acid, preferably a proteinogenic amino acid, more preferably Leu or Thr; b. X2 is selected from Trp, Phe and 2-NaI; c. X3 is an alpha-amino acid, preferably a proteinogenic amino acid, more preferably Leu; d. X4 is Trp; e. X5 is an alpha-amino acid, preferably a proteinogenic amino acid, more preferably Lys; f. X6 is selected from Ala, Trp, D-Leu, D-Thr and D-Glu; g. X7 is Glu; h. X8 is an alpha-amino acid, preferably a proteinogenic amino acid, more preferably Trp; i. X9 is Glu or Thr; j. X10 is an alpha-amino acid, preferably a proteinogenic amino acid, more preferably Lys; k. X11 is selected from Arg, Ser, Cit and hSer; l. X12 is an alpha-amino acid, preferably a proteinogenic amino acid, more preferably Lys or Pra; m. X13 is selected from D-Pro, allylamine, cysteamine and Orn; n. X14 is selected from Pro, Aib or an acid comprising 4-pentenoyl or 3-bromopropionyl moiety; wherein 1) the alpha-amino group of X1 is bound to the a carboxy group of X14; or 2) if X13 is Orn, the alpha-amino group of X1 is bound to the alpha-carboxy group of X13; and the side chain of X13 is bound to the alpha-carboxy group of X12; and wherein said peptide or peptidomimetic assumes a beta-hairpin structure under physiological conditions, wherein X2 to X4 and X9 to X11 are in beta-sheet conformation and/or turns are formed by X5 to X8, and X12 to X14 and X1; (g) X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13-X14, wherein a. X1 is an alpha-amino acid, preferably a proteinogenic amino acid, more preferably Arg or Nmarg; b. X2 is selected from Trp, Glu, 2-NaI and 1-NaI; c. X3 is an alpha-amino acid, preferably a proteinogenic amino acid, more preferably selected from Pro, Trp and Glu; d. X4 is an alpha-amino acid, preferably a proteinogenic amino acid, or preferably selected from Gln, Leu, Arg, and Pra; e. X5 is selected from Lys, Glu, Ser, Lys(N3) and (S)-2-(4′-pentenyl)Ala; f. X6 is Ile or Lys; g. X7 is an alpha-amino acid, preferably a proteinogenic amino acid, or preferably selected from Lys, Trp, 2-NaI and 1-NaI; h. X8 is an alpha-amino acid, preferably a proteinogenic amino acid, more preferably Asp or Glu; i. X9 is selected from Asp, Lys, Ser, (S)-2-(4′-pentenyl)Ala, Pra and D-Pra; j. X10 is Gln; k. X11 is an alpha-amino acid, preferably a proteinogenic amino acid, more preferably Arg or Val; l. X12 is an alpha-amino acid, preferably a proteinogenic amino acid, more preferably Arg or Thr; m. X13 is Arg or Trp; n. X14 is Trp or Arg; wherein X2 to X13 of said peptide or peptidomimetic assume an alpha-helix structure under physiological conditions; and wherein a side chain of X5 is covalently connected to a side chain of X9, wherein a covalent connection may comprise a carbon-carbon double bond; (h) X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13-X14 wherein a. X1 is an alpha-amino acid, preferably a proteinogenic amino acid, more preferably Glu or Nmglu; b. X2 is selected from Trp, 2-NaI, and 1-NaI; c. X3 is Lys, Glu, Ser, Lys(N3) or (S)-2-(4′-pentenyl)Ala; d. X4 is an alpha-amino acid, preferably a proteinogenic amino acid, more preferably selected from Trp, 2-NaI, and 1-NaI; e. X5 is Ile, Trp or Arg; f. X6 is selected from Ile, Leu and Lys; g. X7 is Asp, Lys, Ser, Pra, D-Pra or (S)-2-(4′-pentenyl)Ala; h. X8 is Lys, Glu, Ser, Lys(N3) or (S)-2-(4′-pentenyl)Ala; i. X9 is Ile; j. X10 is Gln; k. X11 is an alpha-amino acid, preferably a proteinogenic amino acid, more preferably Ala or Val; l. X12 is Asp, Lys, Ser, Pra, D-Pra or (S)-2-(4′-pentenyl)Ala; m. X13 is selected from Arg, Lys, and Trp; n. X14 is an alpha-amino acid, preferably a proteinogenic amino acid, more preferably Ala or Trp; wherein a side chain of X3 is covalently connected to a side chain of X7; and wherein a side chain of X8 is covalently connected to a side chain of X12; and wherein X1 to X12 of said peptide or peptidomimetic assume an alpha-helix structure under physiological conditions; or (i) X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13-X14-X15 wherein a. X1 is an alpha-amino acid, preferably a proteinogenic amino acid, more preferably Glu, Arg, Nmglu or Nmarg; b. X2 is selected from Trp, 2-NaI and 1-NaI; c. X3 is an alpha-amino acid, preferably a proteinogenic amino acid, or preferably selected from Trp, 2-NaI, 1-NaI and Glu; d. X4 is Lys, Glu, Ser, Lys(N3) or (S)-2-(4′-pentenyl)Ala; e. X5 is an alpha-amino acid, preferably a proteinogenic amino acid, more preferably Ile or Arg; f. X6 is selected from Ile, Leu and Lys; g. X7 is an alpha-amino acid, preferably a proteinogenic amino acid, more preferably Lys; h. X8 is Asp, Lys, Ser, Pra, D-Pra or (S)-2-(4′-pentenyl)Ala; i. X9 is Ile; j. X10 is Gln; k. X11 is Lys, Glu, Ser, Lys(N3) or (S)-2-(4′-pentenyl)Ala; l. X12 is an alpha-amino acid, preferably a proteinogenic amino acid, more preferably Trp or Ile; m. X13 is selected from Arg, Lys and Trp; n. X14 is an alpha-amino acid, preferably a proteinogenic amino acid, more preferably Arg or Trp; o. X15 is Asp, Lys, Ser, Pra, D-Pra or (S)-2-(4′-pentenyl)Ala; wherein a side chain of X4 is covalently connected to a side chain of X8; and wherein a side chain of X11 is covalently connected to a side chain of X15; and wherein X1 to X15 of said peptide or peptidomimetic assume an alpha-helix structure under physiological conditions.

2. The peptide or peptidomimetic of claim 1, wherein said peptide or peptidomimetic is capable of interfering with ubiquitination.

3. The peptide or peptidomimetic of claim 1, wherein said peptide or peptidomimetic stops cell division or induces cell death.

4. The peptide or peptidomimetic of claim 1, wherein in sequence (a): (a) an alpha-amino group of X1 is bound to a moiety selected from acetyl, benzoyl, 2-acetoxybenzoyl, 3-acetoxybenzoyl, 4-acetoxybenzoyl, Biotin-Ahx, methyl, Ac-Gly-Phe-Trp-Phe-Gly, Ac-Gly-Phe-Trp-Phe-Gly-NH—(CH.sub.2).sub.2—O—CH.sub.2—CO—, Ac-Gly-Phe-Trp-Phe-Gly-NH—(CH.sub.2).sub.2-PEG-O—CH.sub.2—CO—, PEG being polyethylene glycol with a polymerization degree from to 6; and R—COO—(CH.sub.2).sub.n—OCO—, R—COO—(CH.sub.2).sub.n—OCO—N(CH.sub.3)—CH.sub.2—CO—, R—COO—(CH.sub.2).sub.n—OCOO—(CH.sub.2).sub.2—CO—, or R—COO—(CH.sub.2).sub.n—OCO—NH—(CH.sub.2).sub.2—CO—, R being alkyl, cycloalkyl, cycloheteroalkyl, aryl or heteroaryl, alkyl having 1 to 6 carbon atoms, cycles having 4 to 10, preferably 5 or 6 ring atoms and 1 or 2 cycles, R preferably being methyl, ethyl, phenyl or benzyl, and n being 1 or 2; (b) X4 is (R)-2-(7′-octenyl)Ala and X11 is (S)-2-(4′-pentenyl)Ala, and a side chain of X4 is bound to a side chain of X11 by a covalent bond; (c) X4 and X11 are Asp or Glu, and their side chains are bound through a diamine alkyl moiety consisting of —NH—CH.sub.2—(CH.sub.2).sub.n—CH.sub.2—NH—, n being 1, 2 or 3; (d) X4 is D-Cys and X11 is Cys or D-Cys, and a side chain of X4 is bound to a side chain of X11 via an aryl moiety, preferably 1,1′-biphenyl-4,4′-bis(methyl) or 3,3′-bipyridine,6,6′-bis(methyl); (e) X4 and X11 are Cys, and a side chain of X4 is bound to a side chain of X11 via an azoaryl moiety, preferably N,N′-[(1Z)-azodi-4,1-phenylene]diacetamide or cis-3,3′-bis(sulfonato)-4,4′-bis(acetamide)azobenzene; (f) X4 is Dap and X11 is Asp, and a side chain of X4 is bound to a side chain of X11 via CO—CH.sub.2-p-C.sub.6H.sub.4—CH.sub.2—NH—, or —CO—CH.sub.2-p-C.sub.6F.sub.4—CH.sub.2—NH; or (g) X14 is amidated, esterified or bound to a moiety selected from Gly-Phe-Trp-Phe-Gly-NH.sub.2, —NH—(CH.sub.2).sub.2—O—CH.sub.2—CO-Gly-Phe-Trp-Phe-Gly-NH.sub.2 and —NH—(CH.sub.2).sub.2-PEG-O—CH.sub.2—CO-Gly-Phe-Trp-Phe-Gly-NH.sub.2, PEG being polyethylene glycol with a polymerization degree from 1 to 6.

5. The peptide or peptidomimetic of claim 1, wherein in sequence (b): (a) an alpha-amino group of X1 is bound to a moiety selected from acetyl, benzoyl, 2-acetoxybenzoyl, 3-acetoxybenzoyl, 4-acetoxybenzoyl, Biotin-Ahx, methyl, Ac-Gly-Phe-Trp-Phe-Gly, Ac-Gly-Phe-Trp-Phe-Gly-NH—(CH.sub.2).sub.2—O—CH.sub.2—CO—, Ac-Gly-Phe-Trp-Phe-Gly-NH—(CH.sub.2).sub.2-PEG-O—CH.sub.2—CO—, PEG being polyethylene glycol with a polymerization degree from to 6; and R—COO—(CH.sub.2).sub.n—OCO—, R—COO—(CH.sub.2).sub.n—OCO—N(CH.sub.3)—CH.sub.2—CO—, R—COO—(CH.sub.2).sub.n—OCOO—(CH.sub.2).sub.2—CO—, or R—COO—(CH.sub.2).sub.n—OCO—NH—(CH.sub.2).sub.2—CO—, R being alkyl, cycloalkyl, cycloheteroalkyl, aryl or heteroaryl, alkyl having 1 to 6 carbon atoms, cycles having 4 to 10, preferably 5 or 6 ring atoms and 1 or 2 cycles, R preferably being methyl, ethyl, phenyl or benzyl, and n being 1 or 2; (b) wherein if X2 and X11 are Agl or Crt, respectively, a covalent connection may comprise a carbon-carbon double bond; (c) wherein if X2 is Cys and X11 is (S)-2-amino-4-bromobutyric acid, a C—S bond connects X2 with X11; (d) wherein if X2 is Pra and X11 is 2Abu(γ-N3) or Nva(δ-N3), a triazole group connects X2 with X11; (e) wherein if X2 and X11 are Cys, a disulfide bridge connects X2 with X11; (f) wherein if X2 is (S)-2-amino-4-bromobutyric acid and X11 is Cys, a C—S bond connects X2 with X11; (g) wherein if X2 is Hpg and X11 Dap(N3), a triazole group connects X2 with X11 (h) X4 and X9 are independently selected from Trp and Phe; or (i) an alpha-carboxy group of X12 is amidated or bound to a moiety selected from-OEt, —OMe,-NHEt, —NHMe, -Gly-Phe-Trp-Phe-Gly-NH.sub.2, —NH—(CH.sub.2).sub.2—O—CH.sub.2—CO-Gly-Phe-Trp-Phe-Gly-NH.sub.2, and —NH—(CH.sub.2).sub.2-PEG-O—CH.sub.2—CO-Gly-Phe-Trp-Phe-Gly-NH.sub.2, PEG being polyethylene glycol with a polymerization degree from 1 to 6; wherein the connection defined in parts (b) to (g) is implemented by one of the following: ##STR00025## wherein n=1 or 2.

6. The peptide or peptidomimetic of claim 1, wherein in sequence (c): (a) an alpha-amino group of X1 is bound to a moiety selected from acetyl, benzoyl, 2-acetoxybenzoyl, 3-acetoxybenzoyl, 4-acetoxybenzoyl, Biotin-Ahx, methyl, Ac-Gly-Phe-Trp-Phe-Gly, Ac-Gly-Phe-Trp-Phe-Gly-NH—(CH.sub.2).sub.2—O—CH.sub.2—CO—, Ac-Gly-Phe-Trp-Phe-Gly-NH—(CH.sub.2).sub.2-PEG-O—CH.sub.2—CO—, PEG being polyethylene glycol with a polymerization degree from to 6; and R—COO—(CH.sub.2).sub.n—OCO—, R—COO—(CH.sub.2).sub.n—OCO—N(CH.sub.3)—CH.sub.2—CO—, R—COO—(CH.sub.2).sub.n—OCOO—(CH.sub.2).sub.2—CO—, or R—COO—(CH.sub.2).sub.n—OCO—NH—(CH.sub.2).sub.2—CO—, R being alkyl, cycloalkyl, cycloheteroalkyl, aryl or heteroaryl, alkyl having 1 to 6 carbon atoms, cycles having 4 to 10, preferably 5 or 6 ring atoms and 1 or 2 cycles, R preferably being methyl, ethyl, phenyl or benzyl, and n being 1 or 2; (b) X3 and X13 are Cys, and a covalent connection between X3 and X13 comprises a sulfur-sulfur bond; (c) X3 and X13 are Agl, and a covalent connection between X3 and X13 may comprise a carbon-carbon double bond; (d) X3 and X13 are Crt, and a covalent connection between X3 and X13 may comprise a carbon-carbon double bond; (e) X3 is Cys and X13 is (S)-2-amino-4-bromobutyric acid, and a C—S bond connects X3 with X13; (f) X3 is (S)-2-amino-4-bromobutyric acid and X13 is Cys, and a C—S bond connects X3 with X13; (g) X3 is Pra and X13 is 2Abu(γ-N3) or Nva(δ-N3), and a triazole group connects X3 with X13; (h) X3 is Hpg and X13 is Dap(N3), and a triazole group connects X3 with X13; or (i) an alpha-carboxy group of X16 is amidated or bound to a moiety selected from-OEt, —OMe,-NHEt, —NHMe, -Gly-Phe-Trp-Phe-Gly-NH.sub.2, —NH—(CH.sub.2).sub.2—O—CH.sub.2—CO-Gly-Phe-Trp-Phe-Gly-NH.sub.2 and —NH—(CH.sub.2).sub.2-PEG-O—CH.sub.2—CO-Gly-Phe-Trp-Phe-Gly-NH.sub.2, PEG being polyethylene glycol with a polymerization degree from 1 to 6.

7. The peptide or peptidomimetic of claim 1, wherein in sequence (d): (a) X4 and X14 are Cys, and a covalent connection between X4 and X14 comprises a sulfur-sulfur bond; (b) X4 and X14 are Agl, and a covalent connection between X4 and X14 may comprise a carbon-carbon double bond; (c) X4 and X14 are Crt, and a covalent connection between X4 and X14 may comprise a carbon-carbon double bond; (d) X4 is Cys and X14 is (S)-2-amino-4-bromobutyric acid, and a C—S bond connects X4 with X14; (e) X4 is (S)-2-amino-4-bromobutyric acid and X14 is Cys, and a C—S bond connects X4 with X14; (f) X4 is Pra and X14 is 2Abu(γ-N3) or Nva(δ-N3), and a triazole group connects X4 with X14; (g) X4 is Hpg and X14 is Dap(N3), and a triazole group connects X4 with X14; or (h) an alpha-carboxy group of X17 is amidated, esterified or bound to a moiety selected from Gly-Phe-Trp-Phe-Gly-NH.sub.2, —NH—(CH.sub.2).sub.2—O—CH.sub.2—CO-Gly-Phe-Trp-Phe-Gly-NH.sub.2 and —NH—(CH.sub.2).sub.2-PEG-O—CH.sub.2—CO-Gly-Phe-Trp-Phe-Gly-NH.sub.2, PEG being polyethylene glycol with a polymerization degree from 1 to 6.

8. The peptide or peptidomimetic of claim 1, wherein in sequence (e): (a) n alpha-amino group of X1 is bound to a moiety selected from acetyl, benzoyl, 2-acetoxybenzoyl, 3-acetoxybenzoyl, 4-acetoxybenzoyl, Biotin-Ahx, methyl, Ac-Gly-Phe-Trp-Phe-Gly, Ac-Gly-Phe-Trp-Phe-Gly-NH—(CH.sub.2).sub.2—O—CH.sub.2—CO—, Ac-Gly-Phe-Trp-Phe-Gly-NH—(CH.sub.2).sub.2-PEG-O—CH.sub.2—CO—, R—COO—(CH.sub.2).sub.n—OCO—N(CH.sub.3)—CH.sub.2—CO—, R—COO—(CH.sub.2).sub.n—OCOO—(CH.sub.2).sub.2—CO— or R—COO—(CH.sub.2).sub.n—OCO—NH—(CH.sub.2).sub.2—CO—, PEG being polyethylene glycol with a polymerization degree from 1 to 6; and R—COO—(CH.sub.2).sub.n—OCO—, R being alkyl, cycloalkyl, cycloheteroalkyl, aryl or heteroaryl, alkyl having 1 to 6 carbon atoms, cycles having 4 to 10, preferably 5 or 6 ring atoms and 1 or 2 cycles, R preferably being methyl, ethyl, phenyl or benzyl and n being 1 or 2; (b) X1 and X10 are Cys, and a covalent connection between X1 and X10 comprises a sulfur-sulfur bond; (c) X1 and X10 are Agl, and a covalent connection between X1 and X10 may comprise a carbon-carbon double bond; (d) X1 and X10 are Crt, and a covalent connection between X1 and X10 may comprise a carbon-carbon double bond; (e) X1 is Cys and X10 is (S)-2-amino-4-bromobutyric acid, and a C—S bond connects X1 with X10; (f) X1 is (S)-2-amino-4-bromobutyric acid and X10 is Cys, and a C—S bond connects X1 with X10; (g) X1 is Pra and X10 is 2Abu(γ-N3) or Nva(δ-N3), and a triazole group connects X1 with X10; (h) X1 is Hpg and X10 is Dap(N3), and a triazole group connects X1 with X10; or (i) X14 is amidated, esterified or functionalized with Gly-Phe-Trp-Phe-Gly-NH.sub.2, —NH—(CH.sub.2).sub.2—O—CH.sub.2—CO-Gly-Phe-Trp-Phe-Gly-NH.sub.2 and —NH—(CH.sub.2).sub.2-PEG-O—CH.sub.2—CO-Gly-Phe-Trp-Phe-Gly-NH.sub.2, PEG being polyethylene glycol with a polymerization degree from 1 to 6.

9. The peptide or peptidomimetic of claim 1, wherein in sequence (f): (a) X13 is allylamine or cysteamine, wherein an alpha-amino group of X13 is bound to a moiety selected from —CH.sub.2CONH.sub.2, —CH.sub.2CO-Gly-Phe-Trp-Phe-Gly-NH.sub.2, —CH.sub.2COOEt and CH.sub.2COOMe; (b) X14 comprises a 4-pentenoyl, bromoacetyl or 3-bromopropionyl moiety, wherein a carboxy group of said moiety is covalently bound to the alpha-amino group of X1; or (c) wherein a side chain of X13 is covalently connected to a side chain of X14; i. if X13 is allylamine and X14 is 4-pentenoic acid, a covalent connection may comprise a carbon-carbon double bond; ii. if X13 is cysteamine and X14 is 3-bromopropionyl, a carbon-sulfur bond or a sulfur-sulfur bond is comprised in the covalent connection; wherein said covalent connection in accordance with (c) is selected from the following: ##STR00026##

10. The peptide or peptidomimetic of claim 1, wherein in sequence (g): (a) an alpha-amino group of X1 is bound to a moiety selected from acetyl, benzoyl, 2-acetoxybenzoyl, 3-acetoxybenzoyl, 4-acetoxybenzoyl, Biotin-Ahx, methyl, Ac-Gly-Phe-Trp-Phe-Gly, Ac-Gly-Phe-Trp-Phe-Gly-NH—(CH.sub.2).sub.2—O—CH.sub.2—CO—, Ac-Gly-Phe-Trp-Phe-Gly-NH—(CH.sub.2).sub.2-PEG-O—CH.sub.2—CO—, PEG being polyethylene glycol with a polymerization degree from 1 to 6; and R—COO—(CH.sub.2).sub.n—OCO—, R—COO—(CH.sub.2).sub.n—OCO—N(CH.sub.3)—CH.sub.2—CO—, R—COO—(CH.sub.2).sub.n—OCOO—(CH.sub.2).sub.2—CO— or R—COO—(CH.sub.2).sub.n—OCO—NH—(CH.sub.2).sub.2—CO—, R being alkyl, cycloalkyl, cycloheteroalkyl, aryl or heteroaryl, alkyl having 1 to 6 carbon atoms, cycles having 4 to 10, preferably 5 or 6 ring atoms and 1 or 2 cycles, R preferably being methyl, ethyl, phenyl or benzyl and n being 1 or 2; (b) X5 and X9 are Ser, and side chains of X5 and X9 are bound through an adipoyl moiety —CO—(CH.sub.2).sub.4—CO—; or (c) X14 is amidated, esterified or bound to a moiety selected from Gly-Phe-Trp-Phe-Gly-NH.sub.2, —NH—(CH.sub.2).sub.2—O—CH.sub.2—CO-Gly-Phe-Trp-Phe-Gly-NH.sub.2, and —NH—(CH.sub.2).sub.2-PEG-O—CH.sub.2—CO-Gly-Phe-Trp-Phe-Gly-NH.sub.2, PEG being polyethylene glycol with a polymerization degree from 1 to 6.

11. The peptide or peptidomimetic of claim 1, wherein in sequence (h): (a) an alpha-amino group of X1 is bound to a moiety selected from acetyl, benzoyl, 2-acetoxybenzoyl, 3-acetoxybenzoyl, 4-acetoxybenzoyl, Biotin-Ahx, methyl, Ac-Gly-Phe-Trp-Phe-Gly, Ac-Gly-Phe-Trp-Phe-Gly-NH—(CH.sub.2).sub.2—O—CH.sub.2—CO—, Ac-Gly-Phe-Trp-Phe-Gly-NH—(CH.sub.2).sub.2-PEG-O—CH.sub.2—CO—, PEG being polyethylene glycol with a polymerization degree from 1 to 6; and R—COO—(CH.sub.2).sub.n—OCO—, R—COO—(CH.sub.2).sub.n—OCO—N(CH.sub.3)—CH.sub.2—CO—, R—COO—(CH.sub.2).sub.n—OCOO—(CH.sub.2).sub.2—CO— or R—COO—(CH.sub.2).sub.n—OCO—NH—(CH.sub.2).sub.2—CO—, R being alkyl, cycloalkyl, cycloheteroalkyl, aryl or heteroaryl, alkyl having 1 to 6 carbon atoms, cycles having 4 to 10, preferably 5 or 6 ring atoms and 1 or 2 cycles, R preferably being methyl, ethyl, phenyl or benzyl and n being 1 or 2; (b) wherein X3 is Lys and X7 is Asp, or X3 is Glu and X7 is Lys; (c) wherein X8 is Lys and X12 is Asp, or X8 is Glu and X12 is Lys; (d) wherein X3 and X7 are Ser, and side chains of X3 and X7 are bound through an adipoyl moiety —CO—(CH.sub.2).sub.4—CO—; (e) wherein X8 and X12 are Ser, and side chains of X8 and X12 are bound through an adipoyl moiety —CO—(CH.sub.2).sub.4—CO—; or (f) an alpha-carboxy group of X14 is amidated, esterified or bound to a moiety selected from Gly-Phe-Trp-Phe-Gly-NH.sub.2, —NH—(CH.sub.2).sub.2—O—CH.sub.2—CO-Gly-Phe-Trp-Phe-Gly-NH.sub.2 and —NH—(CH.sub.2).sub.2-PEG-O—CH.sub.2—CO-Gly-Phe-Trp-Phe-Gly-NH.sub.2, PEG being polyethylene glycol of different polymerization degrees, preferably from 1 to 6.

12. The peptide or peptidomimetic of claim 1, wherein in sequence (i): (a) an alpha-amino group of X1 is bound to a moiety selected from acetyl, benzoyl, 2-acetoxybenzoyl, 3-acetoxybenzoyl, 4-acetoxybenzoyl, Biotin-Ahx, methyl, Ac-Gly-Phe-Trp-Phe-Gly, Ac-Gly-Phe-Trp-Phe-Gly-NH—(CH.sub.2).sub.2—O—CH.sub.2—CO—, Ac-Gly-Phe-Trp-Phe-Gly-NH—(CH.sub.2).sub.2-PEG-O—CH.sub.2—CO—, PEG being polyethylene glycol with a polymerization degree from 1 to 6; and R—COO—(CH.sub.2).sub.n—OCO—, R—COO—(CH.sub.2).sub.n—OCO—N(CH.sub.3)—CH.sub.2—CO—, R—COO—(CH.sub.2).sub.n—OCOO—(CH.sub.2).sub.2—CO— or R—COO—(CH.sub.2).sub.n—OCO—NH—(CH.sub.2).sub.2—CO—, R being alkyl, cycloalkyl, cycloheteroalkyl, aryl or heteroaryl, alkyl having 1 to 6 carbon atoms, cycles having 4 to 10, preferably 5 or 6 ring atoms and 1 or 2 cycles, R preferably being methyl, ethyl, phenyl or benzyl and n being 1 or 2; (b) wherein X4 is Lys and X8 is Asp, or X4 is Glu and X8 is Lys; (c) wherein X11 is Lys and X15 is Asp, or X11 is Glu and X15 is Lys; (d) wherein X4 and X8 are Ser, and their side chains are bound through the adipoyl moiety —CO—(CH.sub.2).sub.4—CO—; (e) wherein X11 and X15 are Ser, and side chains of X11 and X15 are bound through an adipoyl moiety —CO—(CH.sub.2).sub.4—CO—; or (f) a corresponding carboxy group X15 is amidated, esterified or bound to a moiety selected from Gly-Phe-Trp-Phe-Gly-NH.sub.2, —NH—(CH.sub.2).sub.2—O—CH.sub.2—CO-Gly-Phe-Trp-Phe-Gly-NH.sub.2, and —NH—(CH.sub.2).sub.2-PEG-O—CH.sub.2—CO-Gly-Phe-Trp-Phe-Gly-NH.sub.2, PEG being polyethylene glycol of different polymerization degrees, preferably from 1 to 6.

13. The peptide or peptidomimetic of claim 1, wherein said peptide or peptidomimetic is conjugated via a linker to a ligand of an E3 ligase.

14. (canceled)

15. A medicament comprising one or more of the peptide or peptidomimetic of claim 1.

16. The medicament of claim 15, wherein (a) the one or more peptides or peptidomimetics are the only pharmaceutically active agents comprised in said medicament; (b) said medicament further comprises one or more further pharmaceutically active agents selected from a. agents which interfere with or stop cell division such as TAME, proTAME, apcin, GLMN or Emil; b. agents which induce DNA damage, such as Topoisomerase II inhibitors, preferably etoposide, doxorubicin, and cisplatin; c. agents which interfere with microtubule assembly such as paclitaxel, vincristine, and PLK1 inhibitors including BI6727; d. immunotherapeutic agents such as anti-PD1 antibodies; e. MDM2 inhibitors such as Nutlin; f. histone deacetylase inhibitors such as valproic acid; g. inhibitors of MEK1 and/or MEK2; h. inhibitors of Hsp90 such as 17-allylamino-17-demethoxygeldanamycin (17-AAG); i. BMI-1 inhibitors such as unesbulin (PTC596) and PTC-209; and j. anti-androgen receptor agents; or (c) said medicament is to be administered to a patient which is undergoing, has undergone, or will undergo radiation therapy.

17. A method of treating, ameliorating or curing a hyperproliferative disease and/or an inflammatory disease comprising administering the peptide or peptidomimetic of claim 1.

18. A method of purifying APC/C, said method comprising (a) bringing into contact a sample comprising APC/C with the peptide or peptidomimetic of claim 1; and (b) separating a complex comprising APC/C and said peptide or peptidomimetic from a remainder of constituents of said sample.

19. A method of purifying a RING E3 protein, said method comprising (c) bringing into contact a sample comprising a RING E3 protein with the peptide or peptidomimetic of claim 1; and (d) separating a complex comprising said RING E3 protein and said peptide or peptidomimetic from a remainder of constituents of said sample.

20. A method of detecting APC/C, said method comprising (a) bringing a sample comprising APC/C in contact with the peptide or peptidomimetic of claim 1, wherein said peptide or peptidomimetic carries a detectable label; and (b) detecting a complex comprising APC/C and said peptide or peptidomimetic.

21. A method of detecting a RING E3 protein, said method comprising (c) bringing a sample comprising said RING E3 protein in contact with the peptide or peptidomimetic of claim 1, wherein said peptide or peptidomimetic carries a detectable label; and (d) detecting a complex comprising said RING E3 protein and said peptide or peptidomimetic.

22. A kit comprising or consisting of one or more peptides or peptidomimetics of claim 1.

Description

[1144] The Figures show:

[1145] FIG. 1: Chemical equivalences of functionalities in 3D space among the different scaffolds—Chemical equivalences (resembling similar physicochemical features in 3D space) are depicted with the same symbol.

[1146] FIG. 2: (A) Comparison of the APC/C activity in the presence of 100 μM of molecules of items (b)-(e) of the first aspect (generation 1) or TAME as a previously described APC/C inhibitor. In vitro APC/C ubiquitination assay driven by the E2 enzyme UBE2C was employed to test the APC/C activity, the reaction time was 45 min. A reaction without the APC/C was used as a negative control and as a substrate, fluorophore-labelled securin was used and monitored by SDS-PAGE followed by fluorescence detection.

[1147] (B) The amount of ubiquitinated securin, as shown in (A) was quantified and normalised to the amount of product formed by control (100%) and the mean and SD from 5 independent experiments were plotted. Statistical significance was tested using one-way Anova test, ns (p>0.05); *(p≤0.05); **(p≤0.01); ***(p≤0.001); ****(p≤0.0001).

[1148] FIG. 3: (A) Comparison of the APC/C activity in the presence of 100 μM of molecules of item (f) of the first aspect (generation 2) or G1-3 (SEQ. ID. NO. 3) as the most potent molecule from items (b)-(e) of the first aspect (generation 1). In vitro APC/C ubiquitination assay driven by the E2 enzyme UBE2C was employed to test the APC/C activity, the reaction time was 45 min. Reactions without the APC/C or without the E2 were used as a negative control. As a substrate, fluorophore-labelled securin was used and monitored by SDS-PAGE followed by fluorescence detection. Asterisks indicate truncated forms of securin.

[1149] (B) The amount of ubiquitinated securin, as shown in (A) was quantified and normalized to the amount of product formed by control (100%) and the mean and SD from 3 independent experiments were plotted (left). Analogous quantification of the amount of unmodified securin was done (right), time point 0 min was set to 100%. Statistical significance was tested using one-way Anova test, ns (p>0.05); *(p≤0.05); **(p≤0.01); ***(p≤0.001); ****(p≤0.0001).

[1150] FIG. 4: (A) Comparison of the APC/C activity in the presence of 100 μM of molecules of item (f) of the first aspect (generation 2), including as references G2-4 (SEQ. ID. NO. 17) and G1-3 (SEQ. ID. NO. 3) from item (b) (generation 1) of the first aspect. In vitro APC/C ubiquitination assay driven by the E2 enzyme UBE2C was employed to test the APC/C activity, the reaction time was 45 min. Reactions without the APC/C or without the E2 were used as a negative control, further, a new negative control G2-N2 derived from the inhibitor molecules, was used. Note, G2-6Pra and G2-8 molecules were dissolved in 40% DMSO. As a substrate, fluorophore-labelled securin was used and monitored by SDS-PAGE followed by fluorescence detection. Asterisks indicate truncated forms of securin.

[1151] (B) The amount of ubiquitinated securin, as shown in (A) was quantified and normalized to the amount of product formed by control (100%) and the mean and SD from 3 independent experiments were plotted (left). Analogous quantification of the amount of unmodified securin was done (right), time point 0 min was set to 100%. Statistical significance was tested using one-way Anova test, ns (p>0.05); *(p≤0.05); **(p≤0.01); ***(p≤0.001); ****(p≤0.0001).

[1152] FIG. 5: (A) Comparison of the APC/C activity in the presence of 100 μM of molecules of items (a) and (g) of the first aspect (generation 3), including G2-6 (SEQ. ID. NO. 19) as the most potent molecule from item (f) (generation 2) of the first aspect, G1-3 (SEQ. ID. NO. 3) as the most potent molecule from item (b) of the first aspect and TAME as a previously described APC/C inhibitor. In vitro APC/C ubiquitination assay driven by the E2 enzyme UBE2C was employed to test the APC/C activity, the reaction time was 45 min. Reactions without the APC/C or without the E2 were used as a negative control, further, a negative control G3-N4 derived from the generation 3 molecules was used. As a substrate, fluorophore-labelled securin was used and monitored by SDS-PAGE followed by fluorescence detection. Asterisks indicate truncated forms of securin.

[1153] (B) The amount of ubiquitinated securin, as shown in (A) was quantified and normalized to the amount of product formed by control (100%) and the mean and SD from 3 independent experiments were plotted (left). Analogous quantification of the amount of unmodified securin was done (right), time point 0 min was set to 100%. Statistical significance was tested using one-way Anova test, ns (p>0.05); *(p≤0.05); **(p≤0.01); ***(p≤0.001); ****(p≤0.0001).

[1154] FIG. 6: Comparison of the APC/C activity in vitro in the presence of 100 μM of G3-1 (SEQ. ID. NO. 27), G3-3 (SEQ. ID. NO. 30), G3-4 (SEQ. ID. NO. 31), G3-5 (SEQ. ID. NO. 32), G3-6 (SEQ. ID. NO. 33) and TAME as a previously described APC/C inhibitor. The data represents results of 3 independent in vitro APC/C ubiquitination assay shown in FIG. 5.

[1155] FIG. 7: (A) Comparison of the APC/C activity in the presence of 25, 50, 75 and 100 μM of G3-3 (SEQ. ID. NO. 30), G3-4 (SEQ. ID. NO. 31), G3-5 (SEQ. ID. NO. 32) or G3-6 (SEQ. ID. NO. 33). In vitro APC/C ubiquitination assay driven by the E2 enzyme UBE2C was employed to test the APC/C activity, the reaction time was 45 min. Reactions without the APC/C or without the E2 were used as a negative control, as a substrate, fluorophore-labelled securin was used and monitored by SDS-PAGE followed by fluorescence detection. Asterisks indicate truncated forms of securin.

[1156] (B) The amount of ubiquitinated securin, as shown in (A) was quantified and normalized to the amount of product formed by control (100%) and the mean and SD from 3 independent experiments were plotted (left). Analogous quantification of the amounts of unmodified securin was done (right), time point 0 min was set to 100%. Statistical significance was tested using one-way Anova test, ns (p>0.05); *(p≤0.05); **(p≤0.01); ***(p≤0.001); ****(p≤0.0001).

[1157] FIG. 8: (A) IC.sub.50 determination of the lead molecule G3-6 (SEQ. ID. NO. 33) based on monitoring APC/C activity in the presence of 0.01-100 μM G3-6 (SEQ. ID. NO. 33). In vitro APC/C ubiquitination assay driven by the E2 enzyme UBE2C was employed to test the APC/C activity, the reaction time was 10 min. Reactions without the APC/C or without the E2 were used as a negative control, as a substrate, fluorophore-labelled securin was used and monitored by SDS-PAGE followed by fluorescence detection. Asterisks indicate truncated forms of securin. To prevent ubiquitin chain elongation, methylated ubiquitin was used.

[1158] (B) The amount of ubiquitinated securin (Securin-Ub1, Securin-Ub2 and Securin-Ub3), as shown in (A), was quantified and plotted, different colours of circles represent data from 4 independent experiments. Non-linear curve was fitted and IC.sub.50 of the G3-6 (SEQ. ID. NO. 33) was determined to 1.96 μM.

[1159] FIG. 9: (A) K.sub.M and k.sub.cat determination of the lead molecule G3-6 (SEQ. ID. NO. 33) based on monitoring APC/C activity in the presence or absence of 3 μM G3-6 APC11 inhibitor and different substrate concentrations. In vitro APC/C ubiquitination assay driven by the E2 enzyme UBE2C was employed to monitor the APC/C activity, the reaction time was 2 min. As a substrate 50, 100, 250, 500, 750, 1000, 1250, 2500 and 5000 nM fluorophore-labelled securin was used and monitored by SDS-PAGE followed by fluorescence detection. To prevent ubiquitin chain elongation, methylated ubiquitin was used. Asterisks indicate truncated forms of securin.

[1160] (B) The amount of ubiquitinated securin (Securin-Ub1, Securin-Ub2 and Securin-Ub3), as shown in (A), was quantified and plotted and non-linear curves were fitted. Quantification of 3 independent experiments and a summary of K.sub.M and k.sub.cat are shown. Note, difference between K.sub.M (-G3-6 (SEQ. ID. NO. 33)) and K.sub.M (+G3-6 (SEQ. ID. NO. 33)) is not significant (p=0.0886) using unpaired t test.

[1161] Based on the results of this experiment inhibitory constant (K.sub.i) was calculated using a model for non-competitive inhibition in Prism 6.0. K.sub.i of the lead molecule G3-6 (SEQ. ID. NO. 33) is 5 μM.

[1162] FIG. 10: Comparison of the capability of the lead molecule G3-6 (SEQ. ID. NO. 33) to inhibit in vitro APC/C activity driven by different E2 enzymes, specifically UBE2C, UBE2D and UBE2S. In vitro APC/C ubiquitination assay was employed to test the APC/C activity, G3-6 and G3-N4 molecules were used in 100 μM concentration and the reaction time was 40 min. Reactions without the APC/C or without the E2 were used as a negative control, further, a negative control G3-N4 derived from the generation 3 molecules was used. As a substrate, fluorophore-labelled ubiquitin-cyclin B1 fusion (Ub-cyclin B1) was used and monitored by SDS-PAGE followed by fluorescence detection. The use of the ubiquitin-cyclin B1 fusion allows UBE2S activity to be monitored without the chain-initiating E2 enzymes UBE2S or UBE2D. Representative image from 3 independent experiments is shown.

[1163] FIG. 11: G3-1Pra (SEQ. ID. NO. 28) has the Propargyl (Pra) chemical group that can be employed for click chemistry. G3-1Pra was clicked to azide agarose beads and used for the pull-down in HeLa K cell extract. As a control, empty azide agarose beads, that were treated in the same manner like G3-1Pra beads were used. Another control was magnetic beads coupled with the APC3 antibody or the HA antibody. (A) Western blot analysis was performed and indicated proteins were detected using near-infrared fluorescence imaging. GAPDH serves as a control for unspecific binding. (B) Indicated proteins were quantified. G3-1Pra binds to the APC/C (APC2 and APC11), however, it almost does not bind to Cullin 1, the subunit of E3 ubiquitin ligases related to the APC/C, suggesting G3-1Pra specificity towards the APC/C.

[1164] FIG. 12: G3-6Pra (SEQ. ID. NO. 37) has the Propargyl (Pra) chemical group that can be employed for Click chemistry. G3-6Pra peptides were clicked to azide magnetic beads and used for the pull-down in HeLa K cell extract. As a control, G3-N4Pra negative control molecule was used. Western blot analysis was performed and indicated APC/C subunits were detected using near-infrared fluorescence imaging. GAPDH serves as a control for unspecific binding.

[1165] FIG. 13: G3-6Pra (SEQ. ID. NO. 37) was clicked to azide magnetic beads and used for the pull-down in HeLa K cell extract. As a control, G3-N4Pra negative control molecule was used. Western blot analysis was performed and indicated proteins were detected using near-infrared fluorescence imaging. GAPDH serves as a control for unspecific binding. G3-6Pra molecule binds to the APC/C, however, does not bind to Cullin 1 and RBX1, subunits of E3 ubiquitin ligases related to the APC/C suggesting G3-6Pra specificity towards the APC/C.

[1166] FIG. 14: G3-6_mod1_miniPEG (SEQ. ID. NO. 36) prolongs mitosis by arresting cells in metaphase. HeLa K cells were treated with Ctrl (DMSO) or 50 μM G3-6_mod1_miniPEG (in DMSO) imaged by live bright-field and fluorescence microscopy for 48 h DNA stained by SiR-Hoechst is shown in white. Images show representative control and G3-6_mod1_miniPEG-treated cells. Images are aligned to the entry into mitosis determined by the breakdown of the nuclear envelope (t=0 min).

[1167] FIG. 15: G3-6_mod1_miniPEG (SEQ. ID. NO. 36) prolongs mitosis and causes cell death after metaphase. Left panel: Scatterplots showing the length of mitosis in cells treated and analysed as in FIG. 14. Gray lines indicate the median duration of mitosis. Note, only cells after 10 h of treatment were analysed. Right panel: Cell fate analysis of mitotic cells analysed in the left panel. Cells that required longer than the 75% percentile duration of mitosis are considered as “prolonged”.

[1168] FIG. 16: G2-6 (SEQ. ID. NO. 19) prolongs mitosis and causes cell death. hTERT RPE-1 cells were treated with 100 μM G2-6, G2-N2 or untreated as Ctrl and imaged by bright-field microscopy for 12-24 h Note, G2-N2 is a negative control molecule. (A) Scatterplots showing the length of mitosis. Cells that required longer than the 95% percentile duration of mitosis in Ctrl (30 min) are considered as “prolonged” and are above a dashed line. N indicates total number of analysed cells obtained from 3 independent experiments (in case of G2-N2, 2 independent experiments). (B) Cell fate analysis of mitotic cells analysed in (A).

[1169] FIG. 17: G3-6_mod1_miniPEG molecules (SEQ. ID. NO. 36) prolong mitosis in human cancer cells of different tissue origin (A549, HeLa K, HT-1080, RKO and SW480) by arresting cells in metaphase. Furthermore, G3-6_mod1_miniPEG molecules (SEQ. ID. NO. 36) cause cell death after a metaphase arrest in a subpopulation of HeLa K, HT-1080 and SW480 cells. Cells were treated with DMSO (Ctrl) and 50 μM G3-6_mod1_miniPEG (dissolved in DMSO), and monitored by live cell imaging for 24 h and analysed by single-cell analysis. Note, only cells after 10 h of treatment were analysed. Left panels: box plots (5-95% percentile) showing the length of mitosis in cells treated with DMSO (Ctrl) or 50 μM G3-6_mod1_miniPEG. Black lines indicate median duration of mitosis. Right panels: cell fate analysis of mitotic cells analysed in the left panels. Cells that required longer than one and half median duration of mitosis of Ctrl are considered as “prolonged”. Shown data represent results of two independent experiments with two technical repeats in each experiment. In all cases, 80 or more cells were analysed. Statistical significance was tested using unpaired t test; ns (p>0.05); *(p≤0.05); **(p≤0.01); ***(p≤0.001); ****(p≤0.0001).

[1170] FIG. 18: G1-3Pra (SEQ. ID. NO. 100) has the Propargyl (Pra) chemical group that can be employed for click chemistry. G1-3Pra was clicked to azide agarose beads and used for the pull-down in HeLa K cell extract. As a control (Ctrl), empty azide agarose beads treated in the same manner as G1-3Pra beads were used. Western blot analysis was performed and the indicated proteins were detected using near-infrared fluorescence imaging. GAPDH and CSE1 serve as a control for unspecific binding. Representative scans from three independent experiments are shown.

[1171] FIG. 19: Structural features shared by peptides and peptidomimetics of the invention. Chemical feature equivalences in molecules of claim 1 sharing fold. (A) alpha-Helix, (B) helix-turn and (C) beta-turn. Sequence positions are shown in number boxes. Additional sequence residues along common fold in claim 1 are indicated in full grey boxes. Cross-linkage connections are highlighted with different line types: (______) claim 1(a-d), ( . . . ) claim 1(e) and 1(g), ( - - - ) claim 1(h), ( . . . ) claim 1(i), ( . . . ) claim 1(f). Physicochemical properties of side chain residues involved in protein recognition or used for stabilizing a defined fold are shown with different color and pattern circles. The size and position of the circles represent the level of similarity along the different claim 1 molecules.

[1172] The Examples illustrate the invention.

EXAMPLE 1

Materials and Methods

[1173] Peptides or peptidomimetics were purchased from GenScript Biotech (Netherlands) B. V. with a >95% purity determined by analytical rpHPLC.

General Synthetic Procedure of Preferred Sequences of Items of the First Aspect.

[1174] Peptides or peptidomimetics are preferably prepared by standard Fmoc vs. standard Boc solid-phase synthesis by using Rink Amide MBHA resin for items (a)-(e) and (g)-(i) (Scheme 1), and 2-chlorotritylchloride resin for item (f). Coupling steps involve 4 equiv. of amino acid, 4 equiv. of 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU) as coupling reagent and 5 equiv. of diisopropylethylamine (DIPEA) in DMF. Coupling progress is monitored via Kaiser (ninhydrin) test and coupling of amino acids is repeated when results suggest yields below than 99%. Fmoc deprotections are carried out by 2×1 min treatments with excess (1:1) piperidine: DMF and the resin is sequentially washed with DMF, DCM:MeOH (1:1, v/v), and DCM upon completation. The N-terminus is acetylated by standar methods with Ac.sub.2O for 10 min. The resulting peptides or peptidomimetics are cleaved from resin by treatment with TFA/H.sub.2O/EDT/TIPS (94:2.5:2.5:1) for 2 h followed by precipitation in cold Et.sub.2O. Peptides or peptidomimetics are redisolved in water/acetonitrile and lyophilized. After freeze-drying, the peptides are purified by rpHPLC. Purity of compounds is assessed via analytical rpHPLC (solvent A is 0.065% TFA in 100% water (v/v) and solvent B is 0.05% TFA in 100% acetonitrile (v/v), Table 4).

Additional Synthetic Steps of Preferred Items (a) and (g) of the First Aspect.

[1175] Crosslinking reaction of unnatural olefininc amino acids is carried out by ring-dosing metathesis (RCM) (Scheme 1). The resin is swollen in dry DCM for 30 min and a solution of 4 mg/mL of Grubbs 1.sup.st generation catalyst in dry DCM is added to the resin under inert atmosphere three times for 2 h. The reaction is keeped under inert atmosphere.

Additional Synthetic Steps of Preferred Items (b)-(d) of the First Aspect.

[1176] Peptides or peptidomimetics containing Cys are prepared through Fmoc synthesis by using DIC/Oxyma or DIC/HOBt as carboxyl activators. Disulfide bridge formation can be attempted through the two following strategies. First, the Fmoc-Cys(Mmt)-OH is used. The residue is incorporated in the respective positions following the general synthetic procedure described above. The resin is washed with DCM and treated with 2% TFA in DCM (5×2 min). Cyclization is carried out on-resin using 25 mM solution of N-chlorosuccinimide (NCS) in DMF. In a second strategy, the linear peptides or peptidomimetics are converted to cyclic disulfides by dropwise addition of a saturated solution of I.sub.2 in acetic acid and repurified by rpHPLC.

Additional Synthetic Steps of Preferred Item (f) of the First Aspect.

[1177] Once the linear peptide is fully synthesized, the peptide is cleaved from resin with cold 0.8% TFA in DCM 5 times for one minute. The eluate is treated with DIPEA (1 mL) immediately after TFA treatment. The resin is further washed with DCM and MeOH and the collected solvents in the eluate containing the linear peptide are evaporated in high vacumm. Cyclization is performed by treatment of the resulting crude with 3 equiv. 2-(7-aza-1H-benzotriazole-1-yl)-1,1,3,3,-tetramethyluronium (HATU), 3 equiv. 1-hydroxy-7-azabenzotriazole (HOAt) and 6 equiv. of DIPEA in DMF and stirred for 18 h. Then DMF is removed under high vacuum and the crude is further dissolved in DCM and extracted with 10% acetonitrile in water. After solvent evaporation, the crude peptide is cooled and treated with pre-cooled TFA:H.sub.2O (9:1) for 5 h at 5° C. followed by precipitation in cold Et.sub.2O.

Additional Synthetic Steps of Preferred Item (h) and (i) of the First Aspect.

[1178] Fmoc-Lys(Mtt)-OH and Fmoc-Asp(OPip)-OH are used for lactam cyclization. These residues are incorporated in the respective positions following the general synthetic procedure described above. The resin is washed with DCM and treated with 2% TFA in DCM (5×2 min). Cyclization is carried out on-resin using 2.5 equiv. BOP, 2.5 equiv. DIPEA in DMF 0.5 M.

##STR00024##

TABLE-US-00004 TABLE 4 rpHPLC retention times (R.sub.t) and mass spectrometry data of preferred items (a) and (g) of the first aspect. SEQ. ID. Designation R.sub.t [M + XH].sup.x+ Calculated NO. in the Figures (min) x = 2 x = 3 x = 4 mass 27 G3-1 11.7.sup.a 1017.0 678.3 509.0 2032.20 29 G3-2 15.4.sup.b 951.9 635.0 476.5 1902.27 30 G3-3 17.7.sup.b 995.5 664.0 — 1989.39 31 G3-4 13.8.sup.a 1003.2 669.4 502.2 2004.37 32 G3-5 19.2.sup.b 1003.1 669.0 502.0 2004.37 33 G3-6 21.7.sup.b 1003.0 669.0 502.0 2004.37 .sup.aLinear gradient from 15% to 75% solvent B over 25 min. .sup.bLinear gradient from 5% to 65% solvent B over 25 min.

Antibodies

Primary Antibodies

[1179]

TABLE-US-00005 Antibody target Product no. Company Species Dilution APC11 N/A Moravian mouse WB 1:1000 Biotechnology monoclonal APC11 14090S Cell rabbit WB 1:1000 Signalling monoclonal APC2 12301S Cell rabbit WB 1:1000 Signalling monoclonal APC1 A301-653a Bethyl rabbit WB 1:1000 polyclonal APC8 A301-181A Bethyl rabbit WB 1:1000 polyclonal Cullin 1 32-2400 Thermo Fisher mouse WB 1:1000 Scientific monoclonal RBX1 11922S Cell rabbit WB 1:1000 Signalling monoclonal GAPDH 2118 Cell rabbit WB 1:5000 Signalling polyclonal

Secondary Antibodies

[1180]

TABLE-US-00006 Antibody target Product no. Company Species Dilution anti-rabbit IgG, 926-32213 LI-COR donkey 1:20000 IRDye 800CW Biosciences conjugated antibody anti-mouse IgG, 926-32212 LI-COR donkey 1:20000 IRDye 800CW Biosciences conjugated antibody anti-rabbit IgG, 926-68073 LI-COR donkey 1:20000 IRDye 680RD Biosciences conjugated antibody anti-mouse IgG, 926-68072 LI-COR donkey 1:20000 IRDye 680RD Biosciences conjugated antibody

Cell Lines

[1181] Cells were cultured according to the standard mammalian tissue culture protocol and sterile technique at 37° C. in 5% CO.sub.2 and tested in regular intervals for mycoplasma.

[1182] HeLa K cells were maintained in DMEM (Gibco) supplemented with 10% (v/v) fetal bovine serum (FBS) (Gibco), 1% (v/v) penicillin-streptomycin (Sigma-Aldrich), 1% (v/v) Glutamax (Gibco), and 0.5 μg/ml amphotericin B (Sigma-Aldrich).

[1183] hTERT RPE-1 cells were maintained in DMEM/F12 (Sigma-Aldrich,) supplemented with 10% (v/v) FBS, 1% (v/v) penicillin-streptomycin, 1% (v/v) Glutamax, 0.26% (v/v) sodium bicarbonate (Gibco) and 0.5 μg/mL amphotericin B.

Protein Expression and Purification

APC/C

[1184] The bacmids encoding the human APC/C were a kind gift from David Barford (MRC, Cambridge, UK). SF9 cells (Expression Systems) were co-infected with two recombinant baculoviruses (ratio 2:5; the first corresponding to the virus containing Strep-tagged APC4) encoding the APC/C at a multiplicity of infection (MOI) of ˜1 and a cell density of 1 million cells/ml. SF9 cells were incubated at 27° C. for 72 h. All steps of APC/C purification were performed at 4° C. Cell pellets were re-suspended in APC/C lysis buffer (50 mM Tris-HCl pH 8.3, 250 mM NaCl, 5% Glycerol, 1 mM EDTA, 2 mM DTT, 0.1 mM PMSF, 2 mM Benzamidine, units/ml benzonase, cOmplete protease inhibitor cocktail (Roche)) and disrupted by nitrogen cavitation in a 4639 Cell Disruption Vessel (Parr Instrument Company). Cell extract was cleared by centrifugation at 48 000 g at 4° C. for 1 h. Strep-tagged APC/C was captured on Strep-Tactin Superflow resin (IBA Life Sciences), washed with APC/C wash buffer (50 mM Tris-HCl pH 8,250 mM NaCl, 5% Glycerol, 1 mM EDTA, 2 mM DTT, 2 mM Benzamidine) and eluted with Buffer E (IBA Life Sciences) supplied with additional NaCl (final concentration 250 mM), 2 mM DTT and 2 mM Benzamidine. Peak fractions were concentrated using Vivaspin 6 centrifugal concentrator (VivaProducts) and purified by size-exclusion chromatography (Superose 6 Increase 10/300 GL column; GE healthcare) in APC/C size-exclusion buffer (20 mM Hepes-NaOH pH 8, 200 mM NaCl, 2 mM DTT, 5% Glycerol). Finally, APC/C was flash-frozen in liquid nitrogen and stored at −80° C.

Cdc20

[1185] The bacmid encoding SBP-tagged Cdc20 was a kind gift from Jonathon Pines (ICR, London, UK). SF9 cells were infected with the recombinant baculovirus encoding Cdc20 at a multiplicity of infection (MOI) of ˜1 and a cell density of 1 million cells/ml. SF9 cells were incubated at 27° C. for 72 h. All steps of Cdc20 purification were performed at 4° C. Cell pellets were re-suspended in Cdc20 lysis buffer (250 mM NaCl, 50 mM Tris-HCl pH 8, 1 mM DTT, 5% Glycerol, cOmplete protease inhibitor cocktail, 1 mM PMSF) and disrupted by nitrogen cavitation in a 4639 Cell Disruption Vessel. Cell extract was cleared by centrifugation at 48 000 μg at 4° C. for 1 h. SBP-tagged Cdc20 was captured to Strep-Tactin Superflow resin, washed with 1× Buffer W (IBA Life Sciences) and eluted with Buffer E supplied with additional NaCl (final concentration 250 mM). Finally, glycerol was added to a final concentration of 10% and Cdc20 was flash-frozen in liquid nitrogen and stored at −80° C.

Protein Analysis

SDS-PAGE

[1186] Proteins were separated by SDS-PAGE electrophoresis using precast Bolt 4-12% Bis-Tris Plus protein gels (Thermo Fisher Scientific) and Criterion XT Bis-Tris Midi Protein Gels (Bio-Rad). In different experiments, various conditions were used: in vitro APC/C ubiquitination assays—165 V, 40 min, SDS-MES running buffer (50 mM MES, 50 mM Tris base, 0.1% SDS, 1 mM EDTA, pH 7.3; Thermo Fisher Scientific); in vitro APC/C ubiquitination assays determining K.sub.M—165V, 1 h, SDS-MOPS running buffer (50 mM MOPS, 50 mM Tris base, 0.1% SDS, 1 mM EDTA, pH 7.7; Thermo Fisher Scientific); pull-down binding assay—165 V, 35 min, SDS-MES running buffer.

Western Blot Analysis

[1187] For the protein transfer, the 0.45 μm Immobilon-FL PVDF membrane (Merck Millipore) was used. The membrane was activated in ethanol and then transferred to the Blotting buffer (50 mM SDS-MOPS, 50 mM Tris base, 0.1% SDS, 1 mM EDTA, 20% EtOH). The proteins were transferred using a wet transfer for 1.5 h at 400 mA. Subsequently, the membrane was incubated with the blocking solution, 10% milk in PBS-T (0.02% Tween-20 in PBS), for 1 h at room temperature. To detect proteins of the interest, the membrane was incubated with primary antibodies overnight at 4° C. Followed by washing the membrane 3 times 10 min with PBS-T and incubation with IRDye fluorescently labelled secondary antibodies for 1 h. Prior detection, the membrane was twice washed with PBS-T for 10 min and once with PBS for 10 min. The detection was done using the quantitative near-infrared scanning system Odyssey (LI-COR Biosciences).

In Vitro APC/C Ubiquitination Assay

[1188] APC/C-dependent ubiquitination reactions were performed at 30° C. in 30 mM HEPES pH 7.4, 175 mM NaCl, 8 mM MgCl.sub.2, 0.05% Tween-20, 1 mM DTT and 5% glycerol and contained 20 nM recombinant APC/C (note, for testing the first generation of inhibitors, APC/C and Cdc20 immunoprecipitated from mitotic HeLa K cells was used), 340 nM Cdc20, 46 nM GST-UBA1, 340 nM UBE2C (alternatively 400 nM UBE2D1, 280 nM UBE2S), 21 μM His.sub.6-ubiquitin or if it is indicated 21 μM methylated-ubiquitin (BostonBiochem), 2.6 mM ATP, 10 mM phosphocreatine and 11 μM creatine kinase. As substrates, 35 nM fluorophore-labeled ubiquitin-cyclin B and 50 nM fluorophore-labeled securin were standardly used (fluorophore IRDye 800CW (LI-COR) was used for labelling). In case of K.sub.M and k.sub.cat determination, 50, 100, 250, 500, 750, 1000, 1250, 2500 and 5000 nM fluorophore-labelled securin was used. The reaction was done in the volume of 15 μl, it was quenched after the indicated time with LDS sample buffer (Thermo Fisher Scientific) supplemented with 100 mM DTT and subjected to SDS-PAGE. Detection of fluorescently labelled substrates was done by quantitative near-infrared scanning system Odyssey.

Pull-Down Binding Assay

[1189] HeLa K cell pellet was re-suspended in the extraction buffer (30 mM HEPES pH 7.5, 175 mM NaCl, 2.5 mM MgCl.sub.2, 0.25% NP40, 10% glycerol, 1 mM DTT) supplemented with 10 μM MG132 (VWR), 1 mM PMSF (Sigma-Aldrich), complete protease inhibitor cocktail (Roche) and PhosSTOP phosphatase inhibitors (Roche) and incubated for 20 min on ice, followed by centrifugation of cell debris for 15 min at 4° C. 16 100 g. In mean time, inhibitor molecules containing Propargyl (Pra) chemical group were covalently attached to the azide agarose or azide magnetic resin (Jena Bioscience) by Cu(I)-catalysed azide-alkyne cycloaddition reaction. Specifically, the inhibitor molecules were mixed with azide agarose resin in ratio 0.125 μmol inhibitor/25 μl agarose resin and azide magnetic resin in ratio 0.018 μmol inhibitor/20 μl magnetic resin. The reaction was catalysed by 1 mM CuSO.sub.4, 0.1 mM TBTA (Sigma-Aldrich) and 1 mM Sodium ascorbate and was performed on the rotating wheel for 30 min at room temperature in the volume of 1 ml. Subsequently, the resin was washed 5 times with the extraction buffer. The inhibitor-agarose resin was added to 1 mg of HeLa K cell extract and the inhibitor-magnetic resin was added to 0.5 mg of HeLa K cell extract followed by incubation at 4° C. for 2 h. Followed by washing the resin 5 times with the extraction buffer. Pull-down proteins were eluted by boiling for 15 min with 1×LDS sample buffer supplemented with 100 mM DTT (agarose resin) or by incubation with 1×LDS sample buffer for 10 min at room temperature followed by taking supernatant that was supplemented with 100 mM DTT and boiling it for 10 min. Samples were subjected to SDS PAGE and Western blot analysis.

Statistical Analysis

[1190] Prism 6.0 (Graphpad) was used, unless specified otherwise.

EXAMPLE 2

[1191] Question: Do molecules from items (b)-(e) of the first aspect (generation 1) inhibit APC/C activity in vitro?

[1192] Approach: in vitro APC/C ubiquitination assay

[1193] Conclusion: Molecules of items (b)-(e) of the first aspect effectively inhibit APC/C in vitro. The molecules G1-3 (SEQ. ID. NO. 3) and G1-7 (SEQ. ID. NO. 6) are the most potent and will be used for further development. [FIG. 2]

[1194] Question: Do molecules of item (f) of the first aspect (generation 2) developed based on G1-1 (SEQ. ID. NO. 1), G1-2 (SEQ. ID. NO. 2), G1-3 (SEQ. ID. NO. 3) and G1-4 (SEQ. ID. NO. 48) inhibit APC/C activity in vitro?

[1195] Approach: in vitro APC/C ubiquitination assay

[1196] Conclusion: Molecules of item (f) of the first aspect inhibit APC/C in vitro and the molecule G2-4 (SEQ. ID. NO. 17) is the most potent. [FIG. 3]

[1197] Question: Do optimized molecules of item (f) of the first aspect (generation 2) developed based on G1-1 (SEQ. ID. NO. 1), G1-2 (SEQ. ID. NO. 2), G1-3 (SEQ. ID. NO. 3) and G1-4 (SEQ. ID. NO. 48) inhibit APC/C activity in vitro?

[1198] Approach: in vitro APC/C ubiquitination assay

[1199] Conclusion: The optimized molecules G2-6 (SEQ. ID. NO. 19) and G2-8 (SEQ. ID. NO. 22) inhibit APC/C in vitro the most effectively compared to other molecules of item (f) of the first aspect. [FIG. 4]

[1200] Question: Do molecules of items (a) and (g) of the first aspect (generation 3) developed based on molecules of items (b)-(f) of the first aspect inhibit APC/C activity in vitro?

[1201] Approach: in vitro APC/C ubiquitination assay

[1202] Conclusion: Molecules of items (a) and (g) of the first aspect (generation 3) inhibit APC/C in vitro the most potently compared to previous generations. [FIG. 5]

[1203] Question: Do molecules of items (a) and (g) of the first aspect (generation 3) inhibit APC/C activity more efficiently than TAME?

[1204] Approach: in vitro APC/C ubiquitination assay

[1205] Conclusion: Molecules of items (g) G3-1 (SEQ. ID. NO. 27) and G3-3 (SEQ. ID. NO. 30) and of item (a) G3-4 (SEQ. ID. NO. 31) and G3-6 (SEQ. ID. NO. 33) of the first aspect inhibit APC/C in vitro more efficiently than TAME. [FIG. 6]

[1206] Question: Which molecules from items (a) and (g) of the first aspect (generation 3) inhibit APC/C in vitro the most potently?

[1207] Approach: in vitro APC/C ubiquitination assay, titration of molecules of items (a) and (g) of the first aspect

[1208] Conclusion: G3-6 (SEQ. ID. NO. 33) inhibits APC/C in vitro the most potently and is the lead compound. [FIG. 7]

[1209] Question: What is the IC.sub.50 of the lead molecule G3-6 (SEQ. ID. NO. 33)?

[1210] Approach: in vitro APC/C ubiquitination assay, titration of the G3-6 molecule (SEQ. ID. NO. 33)

[1211] Conclusion: IC.sub.50 of the lead molecule G3-6 (SEQ. ID. NO. 33) is in low micromolar range. [FIG. 8]

[1212] Question: What is the mechanism of action of the G3-6 molecule (SEQ. ID. NO. 33) (type of the inhibition)?

[1213] Approach: in vitro APC/C ubiquitination assay, titration of substrate (securin)

[1214] Conclusion: G3-6 molecule (SEQ. ID. NO. 33) acts as a non-competitive inhibitor of APC/C in vitro. [FIG. 9]

[1215] Question: Is APC/C inhibition by the G3-6 molecule (SEQ. ID. NO. 33) dependent on a specific E2 enzyme?

[1216] Approach: in vitro APC/C ubiquitination assay, different E2 enzymes—UBE2C, UBE2D, UBE2S

[1217] Conclusion: The lead molecule G3-6 (SEQ. ID. NO. 33) inhibits APC/C in vitro independently on employed E2 enzymes. [FIG. 10]

[1218] Question: Is the G3-1 molecule (SEQ. ID. NO. 27) binding specifically to the APC/C?

[1219] Approach: Pull-down binding assay from HeLa K cell extract, azide agarose beads

[1220] Conclusion: The G3-1 molecule (SEQ. ID. NO. 27) binds specifically to the APC/C in HeLa K cell extract. [FIG. 11]

[1221] Question: Is the G3-6 molecule (SEQ. ID. NO. 33) binding to the APC/C?

[1222] Approach: Pull-down binding assay in HeLa K cell extract, azide magnetic beads

[1223] Conclusion: The G3-6 molecule (SEQ. ID. NO. 33) binds to the APC/C in HeLa K cell extract. [FIG. 12]

[1224] Question: Does the G3-6 molecule (SEQ. ID. NO. 33) bind specifically to the APC/C?

[1225] Approach: Pull-down binding assay in HeLa K cell extract, azide magnetic beads

[1226] Conclusion: The G3-6 molecule (SEQ. ID. NO. 33) binds specifically to the APC/C in cell extract. [FIG. 13]

EXAMPLE 3

[1227] Question: What effect has the G3-6_mod1_miniPEG molecule (SEQ. ID. NO. 36) on mitosis of HeLa K cells?

[1228] Approach: Live cell imaging of HeLa K cells in the presence of the G3-6_mod1_miniPEG molecule (SEQ. ID. NO. 36)

[1229] Conclusion: The G3-6_mod1_miniPEG molecule (SEQ. ID. NO. 36) induces a metaphase delay and causes cell death after metaphase in HeLa K cells. [FIG. 14, 15]

[1230] Question: What effect has the G2-6 molecule (SEQ. ID. NO. 19) on mitosis of hTERT RPE-1 cells?

[1231] Approach: Live cell imaging of hTERT RPE-1 cells in the presence of G2-6 (SEQ. ID. NO. 19) and the G2-N2 control molecule

[1232] Conclusion: The G2-6 molecule (SEQ. ID. NO. 19) causes prolonged mitosis and mitotic or subsequent cell death in hTERT RPE-1 cells. [FIG. 16]

Methodology for Example 3

Live Cell Imaging

[1233] Automated time-lapse microscopy was performed using ImageXpress Micro XLS wide-field screening microscope (Molecular Devices) equipped with a 10×, 0.5 NA., 20×, 0.7 NA, and 40×, 0.95 NA Plan Apo air objectives (Nikon), a laser-based autofocus and a full environmental control (5% CO.sub.2, 37° C.). Cells were grown in 96-well plastic bottom plates (clear, Greiner Bio-One) and for live cell imaging media was changed to DMEM without phenol red and riboflavin (Thermo Fisher Scientific), supplemented with 10% (v/v) FBS, 1% (v/v) Glutamax, 1% (v/v) penicillin-streptomycin, and 0.5 μg/ml amphotericin B.

Live Cell Imaging of Cells Treated with Compounds of the Invention

[1234] HeLa K cells were seeded into 96-well plates (4000-4500 cells) one day prior the treatment with compounds of the invention. Cells were treated with 50 μM of compound G3_mod1_mini PEG and cell division was monitored by live cell imaging using ImageXpress Micro XLS wide-field screening microscope, images were acquired every 3 min for time courses of 48 h. The length of mitosis was determined manually.

[1235] hTERT RPE-1 cells were seeded into 96-well plates (5000 cells) one day prior the treatment with compounds of the invention. Cells were treated with 100 μM of compound G2-6 and the negative control molecule G2-N2 and cell division was monitored by live cell imaging using ImageXpress Micro XLS wide-field screening microscope, images were acquired every 3 min for time courses of 12-24 h. The length of mitosis was determined manually.

EXAMPLE 4

[1236] Question: Does the G3-6_mod1_miniPEG molecule (SEQ. ID. NO. 36) prolong mitosis in different human cancer cell lines representing lung carcinoma (A549), cervical carcinoma (HeLa K), fibrosarcoma (HT-1080), colon carcinoma (RKO) and colorectal adenocarcinoma (SW480)?

[1237] Approach: Live cell imaging of A549, HeLa K, HT-1080, RKO and SW480 cells in the presence of the G3-6_mod1_miniPEG molecule (SEQ. ID. NO. 36) and the carrier (DMSO) control.

[1238] Conclusion: The G3-6_mod1_miniPEG molecule (SEQ. ID. NO. 36) prolongs mitosis in human cancer cells of different tissue origin (A549, HeLa K, HT-1080, RKO and SW480) by arresting cells in metaphase. Furthermore, G3-6_mod1_miniPEG molecules (SEQ. ID. NO. 36) cause cell death after a metaphase arrest in a subpopulation of HeLa K, HT-1080 and SW480 cells [FIG. 17]

Methodology for Example 4

Cell Lines

[1239] Cells were cultured according to the standard mammalian tissue culture protocol and sterile technique at 37° C. in 5% CO.sub.2 and tested in regular intervals for mycoplasma.

[1240] A549, HeLa K and SW480 cells were maintained in DMEM (Thermo Fisher Scientific) supplemented with 10% (v/v) fetal bovine serum (FBS) (Thermo Fisher Scientific), 1% (v/v) penicillin-streptomycin (Sigma-Aldrich), and 1% (v/v) Glutamax (Thermo Fisher Scientific).

[1241] HT-1080 and RKO cells were maintained in RPMI 1640 (Thermo Fisher Scientific) supplemented with 10% (v/v) FBS and 1% (v/v) penicillin-streptomycin.

Live Cell Imaging

[1242] Automated time-lapse microscopy was performed using ImageXpress Micro XLS wide-field screening microscope (Molecular Devices) equipped with a 10×, 0.5 NA., 20×, 0.7 NA, and 40×, 0.95 NA Plan Apo air objectives (Nikon), a laser-based autofocus and a full environmental control (5% CO.sub.2, 37° C.). Cells were grown in 96-well plastic bottom plates (clear, Greiner Bio-One) and for live cell imaging media was changed to imaging media. Imaging media for A549, HeLa K and SW480 was DMEM without phenol red and riboflavin (Thermo Fisher Scientific), supplemented with 10% (v/v) FBS, 1% (v/v) Glutamax, and 1% (v/v) penicillin-streptomycin; imaging media for HT-1080 and RKO was RPMI 1640 without phenol red (Thermo Fisher Scientific), supplemented with 10% (v/v) FBS, and 1% (v/v) penicillin-streptomycin.

Live Cell Imaging of Cells Treated with Compounds of the Invention

[1243] A549 (5000-5500 cells), HeLa K (3000-4000 cells), HT-1080 (4000 cells), RKO (5000 cells), and SW480 (7000-8000 cells) cells were seeded into 96-well plates one day prior to treatment with compounds of the invention. Cells were treated with 50 μM of compound G3_mod1_mini PEG and cell division was monitored by live cell imaging using an ImageXpress Micro XLS wide-field screening microscope and images were acquired every 3 min for time courses of 24 h. The length of mitosis was determined manually.

EXAMPLE 5

[1244] Question: Is the G1-3Pra molecule (SEQ. ID. NO. 100) a general RING E3 binder and hence can be used as such general binder or as a template to develop specific and/or optimized RING E3 binders?

[1245] Approach: Pull-down binding assay in HeLa K extract, azide agarose beads and detection of APC/C (APC11) and selected RING ligases involved in carcinogenesis

[1246] Conclusion: The G1-3Pra molecule (SEQ. ID. NO. 100) binds not only to the APC/C (APC11) but also other cancer relevant RING proteins (BMI-1, BRCA1, c-CBL, c-IAP1, MDM2, RBX1, TRAF2) in HeLa K cell extracts.

Methodology for Example 5

Pull-Down Binding Assay

[1247] HeLa K cell pellet was re-suspended in the extraction buffer (30 mM HEPES pH 7.5, 175 mM NaCl, 2.5 mM MgCl2, 0.25% NP40, 10% glycerol, 1 mM DTT supplied with complete protease inhibitor cocktail (Roche), PhosSTOP phosphatase inhibitors (Roche), 1 mM PMSF, 10 μM MG132) and incubated for 20 min on ice, followed by centrifugation to clear the lysate for 15 min at 4° C. 16,100 g. In the meantime, inhibitor molecules containing the propargyl-Gly-OH (Pra) chemical group were covalently attached to the azide agarose resin (Jena Bioscience) by Cu(I)-catalysed azide-alkyne cycloaddition reaction. Specifically, the inhibitor molecules were mixed with azide agarose resin in ratio 0.125 μmol inhibitor/20 μl agarose resin. The reaction was catalysed by 0.1 mM CuSO.sub.4, 0.5 mM THPTA (Sigma-Aldrich) and 5 mM Sodium ascorbate and was performed on the rotating wheel for 30 min at room temperature in the volume of 1 ml. Subsequently, the resin was washed five times with the extraction buffer. The inhibitor-agarose resin was added to 2 mg of HeLa K cell extract and incubated at 4° C. for 2 h. Afterwards, the resin was washed four times with the extraction buffer. Pull-down proteins were eluted by incubation with pre-warmed 1×LDS sample buffer for 10 min at room temperature followed by taking supernatant supplemented with 100 mM DTT and boiling for 10 min. Afterwards the samples were halved and two SDS PAGE and Western blot analyses were performed to detect the indicated proteins.

Primary Antibodies

[1248]

TABLE-US-00007 Antibody target Product no. Company Species Dilution APC11 14090 Cell Rabbit monoclonal WB 1:1000 Signalling BMI1 6964 Cell Rabbit monoclonal WB 1:1000 Signalling BRCA1 9010 Cell Rabbit polyclonal WB 1:1000 Signalling c-CBL 2747 Cell Rabbit polyclonal WB 1:1000 Signalling c-IAP1 7065 Cell Rabbit monoclonal WB 1:1000 Signalling CSE1 AB54674 Abcam Mouse monoclonal WB 1:2500 GAPDH 2118 Cell Rabbit polyclonal WB 1:5000 Signalling MDM2 86934 Cell Rabbit monoclonal WB 1:1000 Signalling RBX1 11922 Cell Rabbit monoclonal WB 1:1000 Signalling TRAF2 4724 Cell Rabbit polyclonal WB 1:1000 Signalling

Secondary Antibodies

[1249]

TABLE-US-00008 Antibody target Product no. Company Species Dilution anti-rabbit IgG, 926-32213 LI-COR donkey 1:20000 IRDye 800CW Biosciences conjugated antibody anti-mouse IgG, 926-68072 LI-COR donkey 1:20000 IRDye 680RD Biosciences conjugated antibody