title: Broad-Spectrum Kinase Profiling in Live Cells with Lysine-Targeted Sulfonyl Fluoride Probes.
abstract: Protein kinases comprise a large family of structurally related enzymes. A major goal in kinase-inhibitor development is to selectively engage the desired kinase while avoiding myriad off-target kinases. However, quantifying inhibitor interactions with multiple endogenous kinases in live cells remains an unmet challenge. Here, we report the design of sulfonyl fluoride probes that covalently label a broad swath of the intracellular kinome with high efficiency. Protein crystallography and mass spectrometry confirmed a chemoselective reaction between the sulfonyl fluoride and a conserved lysine in the ATP binding site. Optimized probe 2 (XO44) covalently modified up to 133 endogenous kinases, efficiently competing with high intracellular concentrations of ATP. We employed probe 2 and label-free mass spectrometry to quantify intracellular kinase engagement by the approved drug, dasatinib. The data revealed saturable dasatinib binding to a small subset of kinase targets at clinically relevant concentrations, highlighting the utility of lysine-targeted sulfonyl fluoride probes in demanding chemoproteomic applications.
authors.forename: Qian
authors.initials: Q
authors.lastname: Zhao
authors.forename: Xiaohu
authors.initials: X
authors.lastname: Ouyang
authors.forename: Xiaobo
authors.initials: X
authors.lastname: Wan
authors.affiliation: Worldwide Research and Development, Pfizer , San Diego, California 92121, United States.
authors.forename: Ketan S
authors.initials: KS
authors.lastname: Gajiwala
authors.affiliation: Worldwide Research and Development, Pfizer , San Diego, California 92121, United States.
authors.forename: John C
authors.initials: JC
authors.lastname: Kath
authors.affiliation: Medicine Design, Pfizer , Cambridge, Massachusetts 02139, United States.
authors.forename: Lyn H
authors.initials: LH
authors.lastname: Jones
authors.forename: Alma L
authors.initials: AL
authors.lastname: Burlingame
authors.forename: Jack
authors.identifier: 0000-0002-9627-5898
authors.initials: J
authors.lastname: Taunton
mesh_terms: D000255:Adenosine Triphosphate / Q000737:chemistry; D001665:Binding Sites; D002478:Cells, Cultured; D000069439:Dasatinib / Q000737:chemistry / Q000494:pharmacology; D016503:Drug Delivery Systems; D008239:Lysine / Q000737:chemistry; D013058:Mass Spectrometry; D008954:Models, Biological; D015335:Molecular Probes / Q000737:chemistry*; D015394:Molecular Structure; D011494:Protein Kinases / Q000737:chemistry*; D013441:Sulfinic Acids / Q000737:chemistry*
chemical_list: D015335:Molecular Probes; D013441:Sulfinic Acids; C048899:sulfuryl fluoride; D000255:Adenosine Triphosphate; D011494:Protein Kinases; D008239:Lysine; D000069439:Dasatinib