Specific inhibitors and active site probes for legumain
Compounds which specifically inhibit legumain, also known as asparaginyl endopeptidase are provided. The compounds have an epoxide or N-Michael acceptor warhead, and have an asparagine side chain attached to a nitrogen atom in the backbone adjacent the warhead. The compounds also preferably comprise a proline residue adjacent the asparagine, and the compound may also contain a third residue and/or a label for cellular or in vivo imaging of active legumain.
1. A compound, having a formula according to either Formula I or Formula II below
wherein,
(a) R1 is either (i) linker-label, wherein linker is lower alkyl, lower alkyl-aryl, or aryl; and label is a radiolabel or an optical label, said linker optionally comprising a cell penetrating peptide; or (ii) lower alkyl, lower alkyl-aryl, or aryl without a label;
(b) P3 is a side chain of leucine or a non-natural amino-acid selected from the group consisting of (2-furyl)alanine, (2-thienyl)alanine, 4-pyridylAla, 1-amino-1-cyclohexane carboxylic acid, 1-amino-1-cyclopentane carboxylic acid, 2-Abz, 3-Abz-, 2-Abu, 3-amino-3-phenyl propionic acid, dehydroAbu, ACPC, Aib, AllylGly, Amb, Amc, Bip, Bpa, Cba, Cha, deltaLeu, deltaVal, Hyp, Igl, Inp, 1-Nal, 2-Nal, Nva, 4-nitroPhe, 4-MethylPhe, 4-Methyl-D Phe, Phe(pI), Phe-4-NH(Boc), hPhe, Phg, pip, Dpip, propargylglycine, Thz, Tic, Tle, 3-NitroTyr, Fmoc-L-neopentylglycine, Fmoc-pCl-L-Phe-OH, Fmoc-pBr-L-Phe-OH, Fmoc-4-amino-tetrahydropyran-4-carboxylic acid, Fmoc-L-Hol, Fmoc-Pip(Boc)-OH, Fmoc-L-styrylalanine, Fmoc-L-homoCha, Fmoc-L-Dab(Boc)-OH, Fmoc-L-Dapa(Boc)-OH, Fmoc-4-[2-(Boc-amino)ethoxy]-L-phenylalanine, and Fmoc-4-(tert-butoxycarbonylmethoxy)-L-phenylalanine;
(c) P2 is selected from the group consisting of (i) proline such that the compound of Formula I is
(ii) a side chain of leucine, and (iii) a side chain of a non-natural amino acid as defined for P3;
(d) W has the formula of a C double bonded to its adjacent CH group or an epoxide group wherein W is C and an epoxide oxygen is bonded to it and an adjacent carbon; and
(e) R2 is selected from the group consisting of lower alkyl, aryl, and lower alkyl-aryl.
2. A compound of claim 1 having the formula,
3. A compound of claim 1 or 2 wherein R1 is lower alkyl linked to a fluorescent dye.
4. A compound of claim 3 wherein the fluorescent dye is Cy5.
5. A compound of claim 2 wherein R1 is linked to a dye and R2 is ethyl.
6. A compound of claim 1 wherein R1 is linked to a dye and R2 is ethyl.
7. A compound of claim 1 according to formula I wherein W is a double bond and P2 is proline.
8. A compound according to one of claim 1 , 2 , 5 or 6 wherein R2 is ethyl, methyl, propyl or butyl.
9. A method of inhibiting legumain activity, comprising the step of contacting the legumain with a compound as defined in claim 1 .
10. A method according to claim 9 wherein R1 is a lower alkyl or aryl group without a label.
11. A method according to claim 9 wherein the compound is a compound having the formula:
12. A method according to claim 9 wherein the compound is a compound having the formula:
13. A method according to claim 12 wherein R1 and R2 are independently a lower alkyl linker having between three and twelve atoms linked to a label.
14. A method according to claim 13 wherein R1 comprises a cell penetrating peptide.
15. A method according to claim 14 wherein R2 is ethyl, methyl, propyl or butyl.
16. A method of imaging a tissue in an organism having active legumain in the tissue, comprising
administering to the organism a compound according to claim 1 wherein R1 is linker-label wherein linker is lower alkyl, lower alkyl-aryl, or aryl; and label is a radiolabel or an optical label, said linker optionally comprising a cell penetrating peptide; and
imaging the tissue of the organism.
17. A method according to claim 16 wherein R1 is a linker to a fluorescent dye.
18. A method according to claim 16 wherein said tissue is a tumor labeled by said compound.
19. A pharmaceutical composition comprising a compound according to claim 1 and an excipient or carrier.
20. The compound of claim 1 , wherein said linker further comprises a cell penetrating peptide selected from the group consisting of a Tat peptide, an arginine-rich permeation peptide sequence based on a Tat peptide, octa-arginine, and penetratin.
21. The compound of claim 20 , wherein the cell penetrating peptide is a Tat peptide.
22. A compound according to claim 4 , wherein R2 is ethyl, methyl, propyl or butyl.