Label free kinase assays and reagents
View Patent ↗This disclosure describes methods and compositions for measuring the binding specificity, kinetics and affinity of kinase inhibitors indirectly using mass sensing analytical techniques, such as SPR, through the competitive displacement of detectable signal-inducing kinase binding molecule. Further provided are methods for preparing such molecules.
1. A method for characterizing a kinase inhibitor candidate using Surface Plasmon Resonance (SPR), comprising:
a) providing a kinase inhibitor candidate;
b) providing a compound of the formula:
[SG] m −[L] n −[KBM],
wherein SG is a signal inducing group; L is a linker; KBM is a kinase binding molecule; m is an integer from 1 to 5; and n is an integer from 0 to 5;
c) providing an immobilized kinase SPR array;
d) contacting the kinase inhibitor candidate and the compound with the immobilized kinase SPR array;
e) measuring the SPR signal of one or both the compound and the kinase inhibitor candidate with the immobilized kinase SPR array; and
f) monitoring a change in the SPR signal upon binding or release of the compound or the kinase inhibitor candidate with the immobilized kinase SPR array, thereby characterizing the kinase inhibitor.
2. The method of claim 1 , wherein the kinase inhibitor candidate is contacted with the immobilized kinase SPR array after the compound is contacted with the immobilized kinase SPR array.
3. The method of claim 1 , wherein the kinase inhibitor candidate is contacted with the immobilized kinase SPR array before the compound is contacted with the immobilized kinase SPR array.
4. The method of claim 1 , wherein the kinase inhibitor candidate and the compound are substantially simultaneously contacted with the immobilized kinase SPR array.
5. The method of claim 1 , wherein the compound is bound to the immobilized kinase SPR array.
6. The method of claim 1 , wherein the kinase inhibitor candidate is bound to the immobilized kinase SPR array.
7. The method of claim 1 , further comprising providing a known kinase inhibitor to characterize the kinase inhibitor candidate.
8. The method of claim 7 , wherein the known kinase inhibitor is bound to the immobilized kinase SPR array.
9. The method of claim 1 , wherein the compound is bound to a binding domain of the kinase in one or more locations on the immobilized kinase SPR array.
10. The method of claim 1 , wherein the kinase inhibitor candidate is bound to a binding domain of the kinase in one or more locations on the immobilized kinase SPR array.
11. The method of claim 1 , further comprising providing a known kinase inhibitor to characterize the kinase inhibitor candidate in one or more locations on the immobilized kinase SPR array.
12. The method of claim 11 , wherein the known kinase inhibitor is bound to a binding domain of the kinase in one or more locations on the immobilized kinase SPR array.