Fluorescent visualization of Src by using dasatinib-BODIPY.
Many biological experiments are not compatible with the use of immunofluorescence, genetically encoded fluorescent tags, or FRET-based reporters. Conjugation of existing kinase inhibitors to cell-permeable fluorophores can provide a generalized approach to develop fluorescent probes of intracellular kinases. Here, we report the development of a small molecule probe of Src through conjugation of BODIPY to two well-established dual Src-Abl kinase inhibitors, dasatinib and saracatinib. We show that this approach is not successful for saracatinib but that dasatinib-BODIPY largely retains the biological activity of its parent compound and can be used to monitor the presence of Src kinase in individual cells by flow cytometry. It can also be used to track the localization of Src by fixed and live-cell fluorescence microscopy. This strategy could enable generation of additional kinase-specific probes useful in systems not amenable to genetic manipulation or could be used together with fluorescent proteins to enable a multiplexed assay readout.
Department of Microbiology and Immunobiology, Harvard Medical School, 77 Avenue Louis Pasteur, Boston, MA 02115 (USA).
Michael L
ML
Vetter
Zijuan
Z
Zhang
Shuai
S
Liu
Jinhua
J
Wang
HaeYeon
H
Cho
Jianming
J
Zhang
Wei
W
Zhang
Nathanael S
NS
Gray
Priscilla L
PL
Yang
D000818:Animals; D052117:Benzodioxoles / Q000737:chemistry / Q000494:pharmacology; D001896:Boron Compounds / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology; D002460:Cell Line; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D000069439:Dasatinib; D004305:Dose-Response Relationship, Drug; D005453:Fluorescence; D006801:Humans; D051379:Mice; D008856:Microscopy, Fluorescence; D015335:Molecular Probes / Q000737:chemistry* / Q000378:metabolism; D015394:Molecular Structure; D047428:Protein Kinase Inhibitors / Q000737:chemistry / Q000494:pharmacology; D011743:Pyrimidines / Q000737:chemistry / Q000494:pharmacology; D011799:Quinazolines / Q000737:chemistry / Q000494:pharmacology; D013329:Structure-Activity Relationship; D013844:Thiazoles / Q000737:chemistry / Q000494:pharmacology; D019061:src-Family Kinases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism
C095489:4,4-difluoro-4-bora-3a,4a-diaza-s-indacene; D052117:Benzodioxoles; D001896:Boron Compounds; D015335:Molecular Probes; D047428:Protein Kinase Inhibitors; D011743:Pyrimidines; D011799:Quinazolines; D013844:Thiazoles; C515233:saracatinib; D019061:src-Family Kinases; D000069439:Dasatinib
cell recognition; fluorescent probes; kinase inhibitors; proteins; signal transduction