Bruton's tyrosine kinase activity probe and method of using
View Patent ↗Described herein are probes for Bruton's tyrosine kinase (Btk). Such probes are used to characterize and develop Btk-selective inhibitors intended for therapeutic use.
1. A Btk activity probe of Formula (I) comprising:
wherein:
A is an irreversible Btk inhibitor moiety having an alkene group which covalently bonds to a cysteine residue of a Btk enzyme having the structure:
wherein L a is O; Ar is substituted or unsubstituted aryl; and Y′ is a 5- or 6-membered heterocycloalkyl;
C is a fluorophore;
B is a linker moiety that links the irreversible Btk inhibitor moiety to the fluorophore;
X is a bond;
Y is selected from the group consisting of a bond, —O(C═O)—, —NR a (C═O)—, —NR a —, —O—, —S—, —S—S—, —O—NR a —, —O(C═O)O—, —O(C═O)NR a —, —NR a (C═O)NR a —, —N═CR a —, —S(C═O)—, —S(O)—, and —S(O) 2 ; and
R a is H or alkyl.
2. The Btk activity probe of claim 1 , wherein the linker moiety covalently links the irreversible Btk inhibitor moiety to the fluorophore.
3. The Btk activity probe of claim 1 , wherein the cysteine residue is in the adenosine triphosphate (ATP) binding pocket of the Btk enzyme.
4. The Btk activity probe of claim 3 , wherein the cysteine residue is Cys 481 of the Btk enzyme.
5. The Btk activity probe of claim 1 , wherein the linker moiety is selected from a bond, an optionally substituted alkyl moiety, an optionally substituted heterocycle moiety, an optionally substituted amide moiety, a ketone moiety, an optionally substituted carbamate moiety, an ester moiety, or a combination thereof.
6. The Btk activity probe of claim 5 , wherein the linker moiety comprises an optionally substituted heterocycle moiety.
7. The Btk activity probe of claim 6 , wherein the optionally substituted heterocycle moiety comprises an optionally substituted piperazinyl moiety.
8. The Btk activity probe of claim 1 , wherein the fluorophore is a 4,4-difluoro-1,3,5,7,8-pentamethyl-4-bora-3a,4a-diaza-s-indacene fluorophore.
9. The Btk activity probe of claim 1 , wherein the fluorophore is a 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid fluorophore.
10. The Btk activity probe of claim 1 , wherein the irreversible Btk inhibitor moiety is:
11. The Btk activity probe of claim 1 having the structure:
12. The Btk activity probe of claim 1 , wherein the probe selectively labels a phosphorylated conformation of Btk.
13. The Btk activity probe of claim 12 , wherein the phosphorylated conformation of Btk is an active form of Btk.
14. The Btk activity probe of claim 1 , wherein the probe is cell permeable.
15. The Btk activity probe of claim 1 , wherein the irreversible Btk inhibitor moiety is:
16. The Btk activity probe of claim 1 having the structure: