Activity-based probe compounds, compositions, and methods of use

Abstract

Activity-based probe compounds for use in labeling a cysteine protease are provided. The compounds are targeted to the protease through a specific targeting element. The compounds additionally include a detectable element, such as a fluorescent label, a radiolabel, or a chelator. In some cases, the compounds additionally include a quenching element that is released upon reaction with the protease. Also provided are compositions comprising the compounds and methods for using the compounds, for example in labeling a protease in an animal and in visualizing a tumor in an animal.

We claim: 
     
         1 - 37 . (canceled) 
     
     
         38 . A method of imaging cells having cysteine protease activity in an animal, comprising:
 administering a pharmaceutically acceptable composition to the animal comprising a compound having the structure of formula (I):   
       
         
           
           
               
               
           
         
         wherein L is 
       
       
         
           
           
               
               
           
         
         L 3  is an optionally substituted alkyl linker, wherein each carbon atom is optionally replaced with a heteroatom; 
         Q is a quencher; 
         T is a targeting element; and 
         D is a fluorescent label having an excitation wavelength and an emission wavelength; 
         permitting the administered composition to interact with a cysteine protease in a tissue of the animal, wherein the interaction causes a cleavage of the composition by the cysteine protease, thereby separating the quencher from the fluorescent label; 
         exposing the tissue of the subject to an excitation signal corresponding to the excitation wavelength of the fluorescent label; 
         detecting an emission signal corresponding to the emission wavelength of the fluorescent label of the fluorescent label, thereby imaging cells having cysteine protease activity, wherein (a) the detected emission signal is associated with a tumor in the animal, (b) the detected emission signal is generated at a tumor margin, and/or (c) the detected emission signal is generated in cells having the cysteine protease activity in the animal. 
       
     
     
         39 . The method of  claim 38 , wherein L is 
       
         
           
           
               
               
           
         
         R is a QSY quencher or QC-1; and 
         n is an integer from 1 to 8. 
       
     
     
         40 . The method of  claim 38 , wherein the fluorescent label is a fluorescein, an Oregon green, a bora-diaza-indecene, a rhodamine, or a cyanine label. 
     
     
         41 . The method of  claim 40 , wherein the fluorescent label is a cyanine label. 
     
     
         42 . The method of  claim 41 , wherein the cyanine label is Cy5. 
     
     
         43 . The method of  claim 38 , wherein T is a peptidic targeting element. 
     
     
         44 . A method of imaging cells having cysteine protease activity in an animal, comprising:
 administering a pharmaceutically acceptable composition to the animal comprising a compound having the structure of formula (II):   
       
         
           
           
               
               
           
         
         wherein L 1  is a linker; 
         AA 1  is an amino acid side chain; 
         U is O, N, or S; 
         R 1  is alkyl, alkenyl, alkynyl, aryl, aralkyl, heteroaryl, heteroaralkyl, cycloalkyl, cycloalkenyl, cycloalkylalkyl, heterocyclyl, heterocyclylalkyl, or a protecting group, and is optionally substituted with 1 to 3 A groups; 
         each A is independently alkyl, alkenyl, alkynyl, alkoxy, alkanoyl, alkylamino, aryl, aryloxy, arylamino, aralkyl, aralkoxy, aralkanoyl, aralkamino, heteroaryl, heteroaryloxy, heteroarylamino, heteroaralkyl, heteroaralkoxy, heteroaralkanoyl, heteroaralkamino, cycloalkyl, cycloalkenyl, cycloalkylalkyl, cycloalkoxy, cycloalkanoyl, cycloalkamino, heterocyclyl, heterocyclyloxy, heterocyclylamino, heterocyclylalkyl, heterocyclylalkoxy, heterocyclylalkanoyl, heterocyclylalkamino, hydroxyl, thio, amino, alkanoylamino, aroylamino, aralkanoylamino, alkylcarboxy, carbonate, carbamate, guanidinyl, urea, halo, trihalomethyl, cyano, nitro, phosphoryl, sulfonyl, sulfonamido, or azido; 
         D is a fluorescent label having an excitation wavelength and an emission wavelength; 
         L 3  is a linker; and 
         Q is a quencher; 
         permitting the administered composition to interact with a cysteine protease in a tissue of the animal, wherein the interaction causes a cleavage of the composition by the cysteine protease, thereby separating the quencher from the fluorescent label; 
         exposing the tissue of the subject to an excitation signal corresponding to the excitation wavelength of the fluorescent label; 
         detecting an emission signal corresponding to the emission wavelength of the fluorescent label of the fluorescent label, thereby imaging cells having cysteine protease activity, wherein (a) the detected emission signal is associated with a tumor in the animal, (b) the detected emission signal is generated at a tumor margin, and/or (c) the detected emission signal is generated in cancer cells having the cysteine protease activity in the animal. 
       
     
     
         45 . The method of  claim 44 , wherein the composition has the structure of formula (III): 
       
         
           
           
               
               
           
         
         wherein R is a QSY or QC-1 quencher; 
         D is a cyanine dye; and 
         m and n are independently integers from 1 to 8. 
       
     
     
         46 . The method of  claim 44 , wherein L 1  is an optionally substituted alkyl linker, wherein each carbon atom is optionally replaced with a heteroatom. 
     
     
         47 . The method of  claim 44 , wherein AA 1  is an aralkyl amino acid side chain, optionally substituted with 1 to 3 A groups. 
     
     
         48 . The method of  claim 44 , wherein U is O. 
     
     
         49 . A method of imaging cells having cysteine protease activity in an animal, comprising:
 administering a pharmaceutically acceptable composition to the animal comprising a compound having the structure of formula (I):   
       
         
           
           
               
               
           
         
         wherein 
         L is L 2 -L 3 -Q; 
         L 2  is a phenoxy group; 
         L 3  is a linker; and 
         Q is a quencher; 
         D-T- is 
       
       
         
           
           
               
               
           
         
         wherein L 1  is a linker; 
         AA 1  is an amino acid side chain; 
         U is O, N, or S; 
         R 1  is alkyl, alkenyl, alkynyl, aryl, aralkyl, heteroaryl, heteroaralkyl, cycloalkyl, cycloalkenyl, cycloalkylalkyl, heterocyclyl, heterocyclylalkyl, or a protecting group, and is optionally substituted with 1 to 3 A groups; and 
         each A is independently alkyl, alkenyl, alkynyl, alkoxy, alkanoyl, alkylamino, aryl, aryloxy, arylamino, aralkyl, aralkoxy, aralkanoyl, aralkamino, heteroaryl, heteroaryloxy, heteroarylamino, heteroaralkyl, heteroaralkoxy, heteroaralkanoyl, heteroaralkamino, cycloalkyl, cycloalkenyl, cycloalkylalkyl, cycloalkoxy, cycloalkanoyl, cycloalkamino, heterocyclyl, heterocyclyloxy, heterocyclylamino, heterocyclylalkyl, heterocyclylalkoxy, heterocyclylalkanoyl, heterocyclylalkamino, hydroxyl, thio, amino, alkanoylamino, aroylamino, aralkanoylamino, alkylcarboxy, carbonate, carbamate, guanidinyl, urea, halo, trihalomethyl, cyano, nitro, phosphoryl, sulfonyl, sulfonamido, or azido; and 
         D is a fluorescent label having an excitation wavelength and an emission wavelength; 
         permitting the administered composition to interact with a cysteine protease in a tissue of the animal, wherein the interaction causes a cleavage of the composition by the cysteine protease, thereby separating the quencher from the fluorescent label; 
         exposing the tissue of the subject to an excitation signal corresponding to the excitation wavelength of the fluorescent label; 
         detecting an emission signal corresponding to the emission wavelength of the fluorescent label of the fluorescent label, thereby imaging cells having cysteine protease activity, wherein (a) the detected emission signal is associated with a tumor in the animal, (b) the detected emission signal is generated at a tumor margin, and/or (c) the detected emission signal is generated in cancer cells having the cysteine protease activity in the animal. 
       
     
     
         50 . The method of  claim 49 , wherein the fluorescent label is a fluorescein, an Oregon green, a bora-diaza-indecene, a rhodamine, or a cyanine label. 
     
     
         51 . The method of  claim 49 , wherein T targets the compound to a cysteine protease. 
     
     
         52 . The method of  claim 49 , wherein L is 
       
         
           
           
               
               
           
         
         wherein each Y is independently an electron-withdrawing group or hydrogen. 
       
     
     
         53 . The method of  claim 51 , wherein L is 
       
         
           
           
               
               
           
         
         R is a QSY or QC-1 quencher; and 
         n is an integer from 1 to 8. 
       
     
     
         54 . A method of imaging cells having cysteine protease activity in an animal, comprising:
 administering a pharmaceutically acceptable composition to the animal comprising a compound having the structure of formula (I):   
       
         
           
           
               
               
           
         
         wherein 
         L is 
       
       
         
           
           
               
               
           
         
         wherein R is an QC-1 quencher and n is an integer from 1 to 8; and 
         D-T- is 
       
       
         
           
           
               
               
           
         
         wherein L 1  is an optionally substituted alkyl linker, wherein each carbon atom is optionally replaced with a heteroatom; 
         AA 1  is an amino acid side chain; 
         U is O; 
         R 1  is alkyl, alkenyl, alkynyl, aryl, aralkyl, heteroaryl, heteroaralkyl, cycloalkyl, cycloalkenyl, cycloalkylalkyl, heterocyclyl, heterocyclylalkyl, or a protecting group, and is optionally substituted with 1 to 3 A groups; and 
         each A is independently alkyl, alkenyl, alkynyl, alkoxy, alkanoyl, alkylamino, aryl, aryloxy, arylamino, aralkyl, aralkoxy, aralkanoyl, aralkamino, heteroaryl, heteroaryloxy, heteroarylamino, heteroaralkyl, heteroaralkoxy, heteroaralkanoyl, heteroaralkamino, cycloalkyl, cycloalkenyl, cycloalkylalkyl, cycloalkoxy, cycloalkanoyl, cycloalkamino, heterocyclyl, heterocyclyloxy, heterocyclylamino, heterocyclylalkyl, heterocyclylalkoxy, heterocyclylalkanoyl, heterocyclylalkamino, hydroxyl, thio, amino, alkanoylamino, aroylamino, aralkanoylamino, alkylcarboxy, carbonate, carbamate, guanidinyl, urea, halo, trihalomethyl, cyano, nitro, phosphoryl, sulfonyl, sulfonamido, or azido; and 
         D is a fluorescent cyanine label having an excitation wavelength and an emission wavelength; 
         permitting the administered composition to interact with a cysteine protease in a tissue of the animal, wherein the interaction causes a cleavage of the composition by the cysteine protease, thereby separating the quencher from the fluorescent label; 
         exposing the tissue of the subject to an excitation signal corresponding to the excitation wavelength of the fluorescent label; 
         detecting an emission signal corresponding to the emission wavelength of the fluorescent label of the fluorescent label, thereby imaging cells having cysteine protease activity, wherein (a) the detected emission signal is associated with a tumor in the animal, (b) the detected emission signal is generated at a tumor margin, and/or (c) the detected emission signal is generated in cancer cells having the cysteine protease activity in the animal. 
       
     
     
         55 . The method of  claim 54 , wherein AA 1  is an aralkyl amino acid side chain, optionally substituted with 1 to 3 A groups.