Fluorescent cyclic peptides, preparation method thereof and use of these peptides for measuring the enzymatic activity of a protease enzyme

1. A cyclic peptide for quantifying the enzymatic activity of an MMP protease enzyme selected from the group consisting of MMP-1, MMP-2, MMP-3, MMP-5, MMP-7, MMP-8, MMP-9, MMP-10, MMP-11, MMP-12, MMP-13, MMP-14, MMP-15, MMP-16, MMP-17, MMP-19, MMP-23, MMP-25 and MMP-26, wherein said cyclic peptide comprises:

, wherein:

S 1 is a first target peptide sequence of an MMP protease enzyme E 1 , wherein S 1 is capable of being recognized and cleaved by said MMP protease enzyme E 1 ;

S 2 is a second target peptide sequence of an MMP protease enzyme E 2 , wherein S 2 is capable of being recognized and cleaved by said MMP protease enzyme E 2 and wherein S 1 and S 2 are the same or different;

E 1 and E 2 correspond to the same MMP protease enzyme;

X 1 is a probe carrying a fluorescent donor group and X 2 is a probe carrying a fluorescent or non-fluorescent acceptor group;

R 1 and R 2 independently correspond to a simple bond, an amino acid residue or a peptide sequence; and

Y is a —CONH— or —NHCO— group;

wherein the MMP protease enzyme is selected from the group consisting of MMP-1, MMP-2, MMP-3, MMP-5, MMP-7, MMP-8, MMP-9, MMP-10, MMP-11, MMP-12, MMP-13, MMP-14, MMP-15, MMP-16, MMP-17, MMP-19, MMP-23, MMP-25 and MMP-26; and

wherein the MMP protease enzyme is MMP-1, and wherein at least one of said first sequence S 1 or the second sequence S 2 , corresponds to at least one of the following sequences:

SEQ. 1:

-Gly-Pro-Gln-Gly-Leu-Leu-Gly-Ala-

SEQ. 2:

-Ala-Pro-Gln-Gly-Ile-Ala-Gly-Gln-

SEQ. 3:

-Gly-Pro-Gln-Gly-Leu-Ala-Gly-Gln-

SEQ. 4:

-Gly-Pro-Leu-Gly-Ile-Ala-Gly-Ile-

SEQ. 5:

-Gly-Pro-Glu-Gly-Leu-Arg-Val-Gly-

SEQ. 6:

-Tyr-Glu-Ala-Gly-Leu-Gly-Val-Val-

SEQ. 7:

-Ala-Gly-Leu-Gly-Val-Val-Glu-Arg-

SEQ. 8:

-Ala-Gly-Leu-Gly-Ile-Ser-Ser-Thr-

SEQ. 9:

-Gly-Ala-Met-Phe-Leu-Glu-Ala-Ile-

SEQ. 10:

-Ile-Pro-Glu-Asn-Phe-Phe-Gly-Val-

SEQ. 11:

-Thr-Glu-Gly-Glu-Ala-Arg-Gly-Ser-

SEQ. 12:

-Arg-Ala-Ile-His-Ile-Gln-Ala-Glu-

SEQ. 13:

-Leu-Arg-Ala-Tyr-Leu-Leu-Pro-Ala-

SEQ. 14:

-Gly-Pro-Leu-Gly-Met-Arg-Gly-Leu-

SEQ. 15:

-Pro-Gln-Gly-Leu-Glu-Ala-Lys-

SEQ. 16:

-Pro-Leu-Gly-Leu-Trp-Ala-D-Arg-

SEQ. 17:

-Pro-Leu-Ala-Leu-Trp-Ala-Arg-

SEQ. 18:

-Pro-Cha-Abu-Cys(Me)-His-Ala-

SEQ. 19:

-Pro-Cha-Gly-Cys(Me)-His-Ala-

SEQ. 20:

-Pro-Cha-Gly-Cys(Me)-His-Ala-

SEQ. 21:

-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-

SEQ. 22:

-Pro-Leu-Gly-[2-mercapto-4-methyl-pentanoyl]-Leu-Gl

SEQ. 23:

-Pro-Leu-Gly-Cys(Me)-His-Ala-D-Arg-

SEQ. 24:

-Arg-Pro-Leu-Ala-Leu-Trp-Arg-

SEQ. 25:

-Pro-Cha-Gly-Nva-His-Ala-

SEQ. 26:

-Arg-Pro-Lys-Pro-Tyr-Ala-Nva-Trp-Met-

wherein the MMP protease enzyme is MMP-2, and wherein at least one of said first sequence S 1 or the second sequence S 2 , corresponds to at least one of the following sequences:

SEQ. 10:

-Ile-Pro-Glu-Asn-Phe-Phe-Gly-Val-

SEQ. 12:

-Arg-Ala-Ile-His-Ile-Gln-Ala-Glu-

SEQ. 13:

-Leu-Arg-Ala-Tyr-Leu-Leu-Pro-Ala-

SEQ. 16:

-Pro-Leu-Gly-Leu-Trp-Ala-D-Arg-

SEQ. 21:

-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-

SEQ. 22:

-Pro-Leu-Gly-[2-mercapto-4-methyl-pentanoyl]-Leu-Gl

SEQ. 23:

-Pro-Leu-Gly-Cys(Me)-His-Ala-D-Arg-

SEQ. 24:

-Arg-Pro-Leu-Ala-Leu-Trp-Arg-

SEQ. 25:

-Pro-Cha-Gly-Nva-His-Ala-

SEQ. 26:

-Arg-Pro-Lys-Pro-Tyr-Ala-Nva-Trp-Met-

SEQ. 27:

Pro-Pro-Gly-Ala-Tyr-His-Gly-Ala-

SEQ. 28

-Gly-Pro-His-Leu-Leu-Val-Glu-Ala-

SEQ. 29:

-Pro-Gln-Gly-Leu-Glu-Ala-Lys-

SEQ. 30:

-Pro-Leu-Ala-Nva-Dpa-Ala-Arg-

SEQ. 31:

-Pro-Leu-Gly-Met-Trp-Ser-Arg-

SEQ. 32:

-Pro-Leu-Gly-SCH[CH 2 CH(CH 3 ) 2 ]-CO-Leu-Gly-

SEQ. 33:

-Arg-Pro-Pro-Gly-Phe-Ser-Ala-Phe-

wherein the MMP protease enzyme is MMP-3, and wherein at least one of said first sequence S 1 or the second sequence S 2 , corresponds to at least one of the following sequences:

SEQ. 5:

-Gly-Pro-Glu-Gly-Leu-Arg-Val-Gly-

SEQ. 10:

-Ile-Pro-Glu-Asn-Phe-Phe-Gly-Val-

SEQ. 12:

-Arg-Ala-Ile-His-Ile-Gln-Ala-Glu-

SEQ. 13:

-Leu-Arg-Ala-Tyr-Leu-Leu-Pro-Ala-

SEQ. 15:

-Pro-Gln-Gly-Leu-Glu-Ala-Lys-

SEQ. 16:

-Pro-Leu-Gly-Leu-Trp-Ala-D-Arg-

SEQ. 20:

-Pro-Cha-Gly-Cys(Me)-His-Ala-

SEQ. 21:

-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-

SEQ. 22:

-Pro-Leu-Gly-[2-mercapto-4-methyl-pentanoyl]-Leu-Gl

SEQ. 26:

-Arg-Pro-Lys-Pro-Tyr-Ala-Nva-Trp-Met-

SEQ. 28

-Gly-Pro-His-Leu-Leu-Val-Glu-Ala-

SEQ. 34:

-Arg-Val-Gly-Phe-Tyr-Glu-Ser-Asp-

SEQ. 35:

-Leu-Leu-Ser-Ala-Leu-Val-Glu-Thr-

SEQ. 36:

-Glu-Ala-Ile-Pro-Met-Ser-Ile-Pro-

SEQ. 37:

-Ile-Ala-Gly-Arg-Ser-Leu-Asn-Pro-

SEQ. 38:

-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-

SEQ. 39

-Asp-Val-Ala-Gln-Phe-Val-Leu-Thr-

SEQ. 40:

-Asp-Thr-Leu-Glu-Val-Met-Arg-Lys-

SEQ. 41:

-Asp-Val-Gly-His-Phe-Arg-Thr-Phe-

SEQ. 42:

-Asp-Ser-Gly-Gly-Phe-Met-Leu-Thr-

SEQ. 43:

-Arg-Val-Ala-Glu-Met-Arg-Gly-Glu-

SEQ. 44:

-Asp-Leu-Gly-Arg-Phe-Gln-Thr-Phe-

SEQ. 45:

-Pro-Phe-Ser-Pro-Leu-Val-Ala-Thr-

SEQ. 46:

-Ala-Pro-Gly-Asn-Ala-Ser-Glu-Ser-

SEQ. 47:

-Phe-Ser-Ser-Glu-Ser-Lys-Arg-Glu-

SEQ. 48:

-Pro-Pro-Glu-Glu-Leu-Lys-Phe-Gln-

SEQ. 49:

-Gly-Pro-Leu-Gly-Met-Arg-Gly-Leu-

SEQ. 50:

-Arg-Pro-Lys-Pro-Val-Glu-Nva-Trp-Arg-Glu-Ala-Lys-

SEQ. 51:

-Pro-Tyr-Ala-Tyr-Trp-Met-Arg-

SEQ. 52:

-Arg-Pro-Lys-Pro-Leu-Ala-Nva-Trp-

wherein the MMP protease enzyme is MMP-5, and wherein said first sequence S 1 or said second sequence S 2 , corresponds to:

SEQ ID NO. 50:

-Arg-Pro-Lys-Pro-Val-Glu-Nva-Trp-Arg-Glu-Ala-Lys-

or

wherein the MMP protease enzyme is MMP-7, and wherein at least one of said first sequence S 1 or the second sequence S 2 , corresponds to at least one of the following sequences:

SEQ. 16:

-Pro-Leu-Gly-Leu-Trp-Ala-D-Arg-

SEQ. 17:

-Pro-Leu-Ala-Leu-Trp-Ala-Arg-

SEQ. 20:

-Pro-Cha-Gly-Cys(Me)-His-Ala-

SEQ. 21:

-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-

SEQ. 22:

-Pro-Leu-Gly-[2-mercapto-4-methyl-pentanoyl]-Leu-Gl

SEQ. 24:

-Arg-Pro-Leu-Ala-Leu-Trp-Arg-

SEQ. 51:

-Pro-Tyr-Ala-Tyr-Trp-Met-Arg-

SEQ. 52:

-Arg-Pro-Lys-Pro-Leu-Ala-Nva-Trp-

wherein the MMP protease enzyme is MMP-8, and wherein at least one of said first sequence S 1 or the second sequence S 2 , corresponds to at least one of the following sequences:

SEQ. 16:

-Pro-Leu-Gly-Leu-Trp-Ala-D-Arg-

SEQ. 17:

-Pro-Leu-Ala-Leu-Trp-Ala-Arg-

SEQ. 20:

-Pro-Cha-Gly-Cys(Me)-His-Ala-

SEQ. 21:

-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-

SEQ. 22:

-Pro-Leu-Gly-[2-mercapto-4-methyl-pentanoyl]-Leu-Gl

SEQ. 23:

-Pro-Leu-Gly-Cys(Me)-His-Ala-D-Arg-

SEQ. 24:

-Arg-Pro-Leu-Ala-Leu-Trp-Arg-

SEQ. 25:

-Pro-Cha-Gly-Nva-His-Ala-

SEQ. 53:

-Pro-Leu-Ala-Tyr-Trp-Ala-Arg-

wherein the MMP protease enzyme is MMP-9, and wherein at least one of said first sequence S 1 or the second sequence S 2 , corresponds to at least one of the following sequences:

SEQ ID NO. 12:

-Arg-Ala-Ile-His-Ile-Gln-Ala-Glu-

SEQ ID NO. 14:

-Gly-Pro-Leu-Gly-Met-Arg-Gly-Leu-

SEQ ID NO. 15:

-Pro-Gln-Gly-Leu-Glu-Ala-Lys-

SEQ ID NO. 16:

-Pro-Leu-Gly-Leu-Trp-Ala-D-Arg-

SEQ ID NO. 19:

-Pro-Cha-Gly-Cys(Me)-His-Ala-

SEQ ID NO. 20:

-Pro-Cha-Gly-Cys(Me)-His-Ala-

SEQ ID NO. 21:

-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-

SEQ ID NO. 22:

-Pro-Leu-Gly42-[2-mercapto-4-methyl-pentanoyl]-

Leu-Gly-

SEQ ID NO. 23:

-Pro-Leu-Gly-Cys(Me)-His-Ala-D-Arg

SEQ ID NO. 32:

-Pro-Leu-Gly-SCH[CH 2 CH(CH 3 ) 2 ]-CO-Leu-Gly-

SEQ ID NO. 33:

-Arg-Pro-Pro-Gly-Phe-Ser-Ala-Phe-

SEQ ID NO. 50:

-Arg-Pro-Lys-Pro-Val-Glu-Nva-Trp-Arg-Glu-Ala-Lys-

SEQ ID NO. 54:

-Gly-Pro-Pro-Gly-Val-Val-Gly-Pro-

SEQ ID NO. 55:

-Gly-Pro-Pro-Gly-Leu-Arg-Gly-Glu-

SEQ ID NO. 56:

-Gly-Pro-Gly-Gly-Val-Val-Gly-Pro-

SEQ ID NO. 57:

-Ile-Pro-Gln-Asn-Phe-Phe-Gly-Val- or

SEQ ID NO. 58:

-Pro-Pro-Gly-Ala-Tyr-His-Gly-Ala- or

wherein the MMP protease enzyme is MMP-10, and wherein at least one of said first sequence S 1 or the second sequence S 2 , corresponds to at least one of the following sequences:

SEQ ID NO. 12:

-Arg-Ala-Ile-His-Ile-Gln-Ala-Glu-

SEQ ID NO. 21:

-Pro-Leu-Gly-Leu-Dpa-Ala-Arg- or

SEQ ID NO. 28:

-Gly-Pro-His-Leu-Leu-Val-Glu-Ala- or

wherein the MMP protease enzyme is MMP-11, and wherein at least one of said first sequence S 1 or the second sequence S 2 , corresponds to at least one of the following sequences:

SEQ ID NO. 20:

-Pro-Cha-Gly-Cys(Me)-His-Ala- or

SEQ ID NO. 21:

-Pro-Leu-Gly-Leu-Dpa-Ala-Arg- or

wherein the MMP protease enzyme is MMP-12, and wherein at least one of said first sequence S 1 or the second sequence S 2 , corresponds to at least one of the following sequences:

SEQ ID NO. 17:

-Pro-Leu-Ala-Leu-Trp-Ala-Arg-

SEQ ID NO. 20:

-Pro-Cha-Gly-Cys(Me)-His-Ala-

SEQ ID NO. 21:

-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-

SEQ ID NO. 22:

-Pro-Leu-Gly-[2-mercapto-4-methyl-pentanoyl]-Leu-

Gly-

SEQ ID NO. 23:

-Pro-Leu-Gly-Cys(Me)-His-Ala-D-Arg-

SEQ ID NO. 24:

-Arg-Pro-Leu-Ala-Leu-Trp-Arg-

SEQ ID NO. 25:

-Pro-Cha-Gly-Nva-His-Ala-

SEQ ID NO. 26:

-Arg-Pro-Lys-Pro-Tyr-Ala-Nva-Trp-Met-

SEQ ID NO. 27:

Pro-Pro-Gly-Ala-Tyr-His-Gly-Ala-

SEQ ID NO. 50:

-Arg-Pro-Lys-Pro-Val-Glu-Nva-Trp-Arg-

SEQ ID NO. 51:

-Pro-Tyr-Ala-Tyr-Trp-Met-Arg-

SEQ ID NO. 52:

-Arg-Pro-Lys-Pro-Leu-Ala-Nva-Trp- or

SEQ ID NO. 59:

-Arg-Pro-Lys-Pro-Gln-Gln-Phe-Trp- or

wherein the MMP protease enzyme is MMP-13, and wherein at least one of said first sequence S 1 or the second sequence S 2 , corresponds to at least one of the following sequences:

SEQ ID NO. 16:

-Pro-Leu-Gly-Leu-Trp-Ala-D-Arg-

SEQ ID NO. 17:

-Pro-Leu-Ala-Leu-Trp-Ala-Arg-

SEQ ID NO. 20:

-Pro-Cha-Gly-Cys(Me)-His-Ala-

SEQ ID NO. 21:

-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-

SEQ ID NO. 22:

-Pro-Leu-Gly-[2-mercapto-4-methyl-pentanoyl]-Leu-

Gly-

SEQ ID NO. 23:

-Pro-Leu-Gly-Cys(Me)-His-Ala-D-Arg-

SEQ ID NO. 24:

-Arg-Pro-Leu-Ala-Leu-Trp-Arg-

SEQ ID NO. 25:

-Pro-Cha-Gly-Nva-His-Ala-

SEQ ID NO. 26:

-Arg-Pro-Lys-Pro-Tyr-Ala-Nva-Trp-Met-

SEQ ID NO. 31:

-Pro-Leu-Gly-Met-Trp-Ser-Arg-

SEQ ID NO. 49:

-Gly-Pro-Leu-Gly-Met-Arg-Gly-Leu-

SEQ ID NO. 50:

-Arg-Pro-Lys-Pro-Val-Glu-Nva-Trp-Arg-

SEQ ID NO. 51:

-Pro-Tyr-Ala-Tyr-Trp-Met-Arg-

SEQ ID NO. 52:

-Arg-Pro-Lys-Pro-Leu-Ala-Nva-Trp-

SEQ ID NO. 53:

-Pro-Leu-Ala-Tyr-Trp-Ala-Arg- or

SEQ ID NO. 59:

-Arg-Pro-Lys-Pro-Gln-Gln-Phe-Trp- or

wherein the MMP protease enzyme is MMP-14, and wherein at least one of said first sequence S 1 or the second sequence S 2 , corresponds to at least one of the following sequences:

SEQ ID NO. 20:

-Pro-Cha-Gly-Cys(Me)-His-Ala-

SEQ ID NO. 21:

-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-

SEQ ID NO. 22:

-Pro-Leu-Gly-[2-mercapto-4-methyl-pentanoyl]-Leu-

Gly- or

SEQ ID NO. 60:

-Pro-Leu-Ala-Cys(p-OMeBz)-Trp-Ala-Arg- or

wherein the MMP protease enzyme is MMP-15, and wherein said first sequence S 1 or the second sequence S 2 , corresponds to the sequence:

SEQ ID NO. 21:

-Pro-Leu-Gly-Leu-Dpa-Ala-Arg- or

wherein the MMP protease enzyme is MMP-16, and wherein said first sequence S 1 or the second sequence S 2 , corresponds to the sequence:

SEQ ID NO. 21:

-Pro-Leu-Gly-Leu-Dpa-Ala-Arg- or

wherein the MMP protease enzyme is MMP-17, and wherein at least one of said first sequence S 1 or the second sequence S 2 , corresponds to at least one of the following sequences:

SEQ ID NO. 21:

-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-

SEQ ID NO. 22:

-Pro-Leu-Gly-[2-mercapto-4-methyl-pentanoyl]-Leu-

Gly- or

wherein the MMP protease enzyme is MMP-19, and wherein said first sequence S 1 or the second sequence S 2 , corresponds to the sequence:

SEQ ID NO. 21:

-Pro-Leu-Gly-Leu-Dpa-Ala-Arg- or

wherein the MMP protease enzyme is MMP-23, and wherein at least one of said first sequence S 1 or the second sequence S 2 , corresponds to the sequence:

SEQ ID NO. 21:

-Pro-Leu-Gly-Leu-Dpa-Ala-Arg- or

wherein the MMP protease enzyme is MMP-25, and wherein at least one of said first sequence S 1 or the second sequence S 2 , corresponds to the sequence:

SEQ ID NO. 21:

-Pro-Leu-Gly-Leu-Dpa-Ala-Arg- or

wherein the MMP protease enzyme is MMP-26, and wherein at least one of said first sequence S 1 or the second sequence S 2 , corresponds to the sequence:

SEQ ID NO. 16:

-Pro-Leu-Gly-Leu-Trp-Ala-D-Arg-.

2. The cyclic peptide according to claim 1 , wherein the donor group carried by the X 1 probe and the acceptor group carried by the X 2 probe comprise an aromatic nucleus.

3. The cyclic peptide according to claim 2 , wherein the donor group carried by the X 1 probe and the acceptor group carried by the X 2 probe comprise an aromatic nucleus and wherein said aromatic nucleus is selected from the group consisting of a benzene, anthracene and coumarin nucleus.

4. The cyclic peptide according to claim 1 , wherein the donor group carried by the X 1 probe and the acceptor group carried by the X 2 probe are selected from the group of pairs consisting of: Tryptophan/2,4-dinitrophenyl; o-aminobenzoic acid/2,4-dinitrophenyl; (7-methoxycoumarin-4-yl)-acetyl/2,4-dinitrophenyl; (7-methoxycoumarin-4-yl)-acetyl/N-3-(2,4-dinitrophenyl)-L-2,3-diaminopropyl; Tryptophan/Dansyl; N-methylanthranoyl/2,4-dinitrophenyl; 6,7-dimethoxycoumarin-4-yl-acetyl/6-N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)aminohexanoic acid; 5-(2′-aminomethyl)naphtalene sulphonic acid/4-(4′-dimethylaminophenylaza)benzoic acid; and ethoxycoumarin-3-carboxylic acid/7-diethylaminocoumarin-3-carboxylic acid.

5. The cyclic peptide according to claim 1 , wherein the donor group carried by the X 1 probe and the acceptor group carried by the X 2 probe comprise a coumarin nucleus.

6. The cyclic peptide according to claim 1 , wherein the donor group carried by the X 1 probe and the acceptor group carried by the X 2 probe respectively correspond to the following formulas:

7. The cyclic peptide according to claim 1 , wherein the probes X 1 and X 2 respectively meet the following formulas:

8. The cyclic peptide of claim 1 , the peptide sequences S 1 and S 2 comprise the formula:

SEQ ID NO. 1:

-Gly-Pro-Gln-Gly-Leu-Leu-Gly-Ala-.

9. The cyclic peptide of claim 8 , comprising the formula:

R 1 , Y, X 1 , X 2 , and R 2 meeting the same definition as the definition given in claim 1 .

10. The cyclic peptide of claim 9 , wherein R 1 is a simple bond, R 2 is Lys, X 1 is Lys(DAC), and X 2 is Lys(MC).

11. A reagent comprising at least one cyclic peptide of claim 1 .

12. A kit comprising:

a first compartment comprising at least one cyclic peptide of claim 1 ; and

a second compartment comprising a buffer solution.