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.
Carrie M
CM
Gower
Novartis Institutes for Biomedical Research , Cambridge, Massachusetts 02139, United States.
Jason R
JR
Thomas
Novartis Institutes for Biomedical Research , Cambridge, Massachusetts 02139, United States.
Edmund
E
Harrington
Novartis Institutes for Biomedical Research , Cambridge, Massachusetts 02139, United States.
Jason
J
Murphy
Matthew E K
ME
Chang
Novartis Institutes for Biomedical Research , Cambridge, Massachusetts 02139, United States.
Ivan
I
Cornella-Taracido
Novartis Institutes for Biomedical Research , Cambridge, Massachusetts 02139, United States.
Rishi K
RK
Jain
Novartis Institutes for Biomedical Research , Cambridge, Massachusetts 02139, United States.
Markus
M
Schirle
Dustin J
DJ
Maly
D001665:Binding Sites; D004789:Enzyme Activation / Q000187:drug effects; D016044:Fusion Proteins, bcr-abl / Q000037:antagonists & inhibitors* / Q000378:metabolism; D006801:Humans; D020128:Inhibitory Concentration 50; D020014:K562 Cells; D019950:Mitogen-Activated Protein Kinase 1 / Q000037:antagonists & inhibitors; D008958:Models, Molecular; D015394:Molecular Structure; D010766:Phosphorylation; D047428:Protein Kinase Inhibitors / Q000737:chemistry* / Q000494:pharmacology; D040901:Proteomics
D047428:Protein Kinase Inhibitors; D016044:Fusion Proteins, bcr-abl; C535150:MAPK1 protein, human; D019950:Mitogen-Activated Protein Kinase 1