The present invention relates to molecular probes of the formula (I) {L1-R1-L} n -A-CO—NH—R2-L2 (I) as defined herein that allow for the observation of the catalytic activity of a selected cathepsin in in vitro assays, in cells or in multicellular organisms, a method for their preparation and the use thereof.
1. A molecular probe for cathepsin protease S of the formula (I):
wherein
X is —CONH—R2-L2
Y is-L-R1-L1, and
R1 and R2 are, independently of each other, each a linker;
L is a bond or a group allowing for a facile conjugation of the group L1;
L1 and L2 are, independent of each other, at least one label optionally bound to a solid support.
2. The molecular probe according to claim 1 , wherein L is a direct bond or a group selected from
—(NRx)-, —O—, —C═N—, —C(═O)—, —C(═O)—NH—, —NH—C(═O)—, —C(═O)H, —CRx=Cry—, —C≡C— and phenyl, wherein Rx and Ry are independently H or (C 1 -C 6 )alkyl.
3. The molecular probe according to claim 1 , wherein R1 or R2 is a straight or branched chain alkylene group with 1 to 300 carbon atoms, wherein optionally;
(a) one or more carbon atoms are replaced by oxygen;
(b) one or more carbon atoms are replaced by nitrogen carrying a hydrogen atom, and the adjacent carbon atoms are substituted by oxo, representing an amide function —NH—CO—;
(c) one or more carbon atoms are replaced by an ester function —O—CO—;
(d) the bond between two adjacent carbon atoms is a double or a triple bond; and/or
(e) two adjacent carbon atoms are replaced by a disulfide linkage.
4. The molecular probe according to claim 3 , wherein labels L1 and L2 are independently of each other a spectroscopic probe; a fluorophore; a quencher or a chromophore; a magnetic probe; a contrast reagent; a molecule which is one part of a specific binding pair which is capable of specifically binding to a partner; a molecule covalently attached to a solid support; a biomolecule; or a positively charged linear or branched polymer.
5. The molecular probe according to claim 4 , wherein labels L1 and L2 are independently of each other bound to a positively charged linear or branched polymer.
6. The molecular probe according to claim 5 , wherein one of labels L1 and L2 is a linear poly(arginine) of D- and/or L-arginine with 6-15 arginine residues.
7. The molecular probe according to claim 4 , wherein L1 is one member and L2 is the other member of two interacting spectroscopic probes L1/L2.
8. The molecular probe according to claim 7 , wherein L1/L2 is a FRET pair.
9. The molecular probe according to claim 8 , wherein one of labels L1 and L2 is a fluorophore selected from Alexa 350, dimethylaminocoumarin, 5/6-carboxyfluorescein, Alexa 488, ATTO 488, DY-505, 5/6-carboxyfluorescein, Alexa 488, Alexa 532, Alexa 546, Alexa 555, ATTO 488, ATTO 532, tetramethylrhodamine, Cy 3, DY-505, DY-547, Alexa 635, Alexa 647, ATTO 600, ATTO 655, DY-632, Cy 5, DY-647 and Cy5.5, and the other of labels L1 and L2 is a quencher selected from Dabsyl, Dabcyl, BHQ 1, QSY 35, BHQ 2, QSY 9, ATTO 540Q, BHQ 3, ATTO 612Q, and QSY 21.
10. A method of using a probe according to claim 1 for imaging a living organism, said method comprising:
(a) administering to said organism a probe of the formula (I);
(b) exposing said organism to electromagnetic radiation which excites non-quenched fluorophore to produce a detectible signal; and
(c) detecting said signal and creating an image thereby.
11. A method of using a probe according to claim 1 for imaging a living organism, said method comprising:
a) administering to said organism a probe of the formula (I);
(b) exposing said organism to electromagnetic radiation which excites fluorophore to produce a detectible signal; and
(c) detecting said signal and creating an image thereby.
12. The molecular probe according to claim 3 , wherein every third carbon atom of said straight or branched chain alkylene group is replaced by oxygen.
13. The molecular probe according to claim 12 , wherein said straight or branched chain alkylene group comprises a poylethyleneoxy group with 1 to 100 ethyleneoxy units.
14. The molecular probe according to claim 4 , wherein said solid support is a glass slide, a microtiter plate or a polymer.
15. The probe according to claim 1 , selected from the group consisting of