Synthetic substrates for enzymes that catalyze reactions of modified cysteines and related methods
Synthetic probes for detecting the activity of enzymes that catalyze reactions of post-translationally modified cysteine residues are described. The probes include “turn-on” probes that include a carbamate linkage that is cleaved via an intramolecular reaction with a free thiol produced by an enzyme catalyzed activity. The probes also include ratiometric, Michael addition-based probes that respond to enzymatic activity by a change in structure that results in a change in fluorescence properties. Methods of using the probes to detect enzymatic activity and disease are described. For example, the probes can be used to detect enzymatic activity in a variety of samples, including live cells and heterogeneous tissues. In addition, prodrugs that can be activated by enzymes that catalyze reactions of post-translationally modified cysteine residues and methods of using the prodrugs to treat disease are described.
1. A compound having a structure of Formula (Ia):
wherein:
A is a residue of a compound A-OH, wherein A-OH is a fluorescent compound, a bio-luminescent compound, or a magnetic resonance imaging (MRI)-detectable compound, wherein said fluorescent compound is selected from the group consisting of a xanthene derivative, a rhodol derivative, and a fluorescein derivative;
R is selected from the group consisting of H, alkyl, substituted alkyl, aralkyl, substituted aralkyl, aryl, and substituted aryl; and
A′ is selected from the group consisting of:
wherein:
R 1 is selected from the group consisting of H, alkyl, substituted alkyl, aralkyl, substituted aralkyl, aryl, and substituted aryl; and
R 2 is selected from the group consisting of H, alkyl, substituted alkyl, aralkyl, substituted aralkyl, aryl, and substituted aryl; or
wherein R 1 and R 2 together with the nitrogen atom to which they are attached form an amino acid or peptidyl residue;
R 3 is selected from the group consisting of alkyl, substituted alkyl, aralkyl, substituted aralkyl, aryl, substituted aryl, S(═O)R 5 , —SH, —N(═O), S-glutathione, and —C(═O)R 5 ;
R 4 is selected from H and R 5 ; and
R 5 is selected from the group consisting of alkyl, substituted alkyl, aralkyl, and substituted aralkyl; or a
pharmaceutically acceptable salt thereof.
2. The compound of claim 1 , wherein A′ is
wherein R 1 , R 2 , and R 3 are as defined in claim 1 .
3. The compound of claim 2 , wherein the compound has a structure of Formula (IIa):
4. The compound of claim 3 , wherein R 5 is —(CH 2 ) n CH 3 wherein n is 1-18.
5. The compound of claim 1 , wherein R 1 and R 2 together with the nitrogen atom to which they are attached form an amino acid or peptidyl residue and further wherein the amino acid or peptidyl residue comprises a positively charged amino acid residue.
6. The compound of claim 1 , wherein the compound has a structure of Formula (Ia) wherein A is a residue of a compound A-OH, wherein A-OH is a or fluorescent compound and wherein the OH of the compound A-OH is a phenolic group.
7. A compound having a structure:
wherein:
A is a residue of a compound A-OH, wherein A-OH is a rhodol or a fluorescein derivative;
R is selected from the group consisting of H, alkyl, substituted alkyl, aralkyl, substituted aralkyl, aryl, and substituted aryl; and
A′ is selected from the group consisting of:
wherein:
R 1 is selected from the group consisting of H, alkyl, substituted alkyl, aralkyl, substituted aralkyl, aryl, and substituted aryl; and
R 2 is selected from the group consisting of H, alkyl, substituted alkyl, aralkyl, substituted aralkyl, aryl, and substituted aryl; or
wherein R 1 and R 2 together with the nitrogen atom to which they are attached form an amino acid or peptidyl residue;
R 3 is selected from the group consisting of alkyl, substituted alkyl, aralkyl, substituted aralkyl, aryl, substituted aryl, —S(═O)R 5 , —SH, —N(═O), S-glutathione, and —C(═O)R 5 ;
R 4 is selected from H and R 5 ; and
R 5 is selected from the group consisting of alkyl, substituted alkyl, aralkyl, and substituted aralkyl; or a
pharmaceutically acceptable salt thereof.
8. The compound of claim 1 , wherein the compound is selected from the group consisting of:
9. A method of detecting enzymatic activity in a sample, the method comprising:
contacting the sample with a compound of Formula (Ia):
wherein:
A is a residue of a compound A-OH, wherein A-OH is a fluorescent compound selected from the group consisting of a xanthene derivative, a rhodol derivative, and a fluorescein derivative;
R is selected from the group consisting of H, alkyl, substituted alkyl, aralkyl, substituted aralkyl, aryl, and substituted aryl; and
A′ is selected from the group consisting of:
wherein:
R 1 is selected from the group consisting of H, alkyl, substituted alkyl, aralkyl, substituted aralkyl, aryl, and substituted aryl; and
R 2 is selected from the group consisting of H, alkyl, substituted alkyl, aralkyl, substituted aralkyl, aryl, and substituted aryl; or
wherein R 1 and R 2 together with the nitrogen atom to which they are attached form an amino acid or peptidyl residue;
R 3 is selected from the group consisting of alkyl, substituted alkyl, aralkyl, substituted aralkyl, aryl, substituted aryl, —S(═O)R 5 , —SH, —N(═O), S-glutathione, and —C(═O)R 5 ;
R 4 is selected from H and R 5 ; and
R 5 is selected from the group consisting of alkyl, substituted alkyl, aralkyl, and substituted aralkyl; or a
pharmaceutically acceptable salt thereof; and
detecting one or more fluorescence signals in the sample, wherein detecting the one or more fluorescence signals detects the presence and/or amount of enzymatic activity of an enzyme in the sample that catalyzes a reaction of a modified cysteine substrate.
10. The method of claim 9 , wherein the enzyme is a thioesterase, an abhydrolyase domain containing 17A (ABHD17) protein, or a thioetherase.
11. The method of claim 9 , wherein the sample is an in vitro sample.
12. The method of claim 9 , wherein the sample comprises a live cell, tissue, or organ.
13. The method of claim 9 , wherein the sample is a subject or a cell, tissue, or fluid obtained from a subject and wherein detecting enzymatic activity further detects a disease state associated with increased or decreased activity of an enzyme that catalyzes a reaction of a modified cysteine substrate.