Conversion of a Single Polypharmacological Agent into Selective Bivalent Inhibitors of Intracellular Kinase Activity

Loss-of-function
studies are valuable for elucidating kinase function
and the validation of new drug targets. While genetic techniques,
such as RNAi and genetic knockouts, are highly specific and easy to
implement, in many cases post-translational perturbation of kinase
activity, specifically pharmacological inhibition, is preferable.
However, due to the high degree of structural similarity between kinase
active sites and the large size of the kinome, identification of pharmacological
agents that are sufficiently selective to probe the function of a
specific kinase of interest is challenging, and there is currently
no systematic method for accomplishing this goal. Here, we present
a modular chemical genetic strategy that uses antibody mimetics as
highly selective targeting components of bivalent kinase inhibitors.
We demonstrate that it is possible to confer high kinase selectivity
to a promiscuous ATP-competitive inhibitor by tethering it to an antibody
mimetic fused to the self-labeling protein SNAPtag. With this approach,
a potent bivalent inhibitor of the tyrosine kinase Abl was generated.
Profiling in complex cell lysates, with competition-based quantitative
chemical proteomics, revealed that this bivalent inhibitor possesses
greatly enhanced selectivity for its target, BCR-Abl, in K562 cells.
Importantly, we show that both components of the bivalent inhibitor
can be assembled in K562 cells to block the ability of BCR-Abl to
phosphorylate a direct cellular substrate. Finally, we demonstrate
the generality of using antibody mimetics as components of bivalent
inhibitors by generating a reagent that is selective for the activated
state of the serine/threonine kinase ERK2.