Journal
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
Volume 109, Issue 24, Pages 9402-9407Publisher
NATL ACAD SCIENCES
DOI: 10.1073/pnas.1202786109
Keywords
interactom profile; azidation; photoactivation
Categories
Funding
- Hungarian Academy of Sciences, HAS-ELTE research group [01055]
- Norway Grant [NNF2-85613]
- Hungarian Scientific Research Fund [K71915]
- Hungarian Academy of Sciences [LP2011-006/2011]
- European Research Council (European Community) [208319]
- National Development Agency [NTP TECH_08_A1/2-2008-0106]
- European Union
- European Social Fund [TAMOP 4.2.1/B-09/1/KMR-2010-0003]
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Photoreactive compounds are important tools in life sciences that allow precisely timed covalent crosslinking of ligands and targets. Using a unique technique we have synthesized azidoblebbistatin, which is a derivative of blebbistatin, the most widely used myosin inhibitor. Without UV irradiation azidoblebbistatin exhibits identical inhibitory properties to those of blebbistatin. Using UV irradiation, azidoblebbistatin can be covalently crosslinked to myosin, which greatly enhances its in vitro and in vivo effectiveness. Photo-crosslinking also eliminates limitations associated with the relatively low myosin affinity and water solubility of blebbistatin. The wavelength used for photo-crosslinking is not toxic for cells and tissues, which confers a great advantage in in vivo tests. Because the crosslink results in an irreversible association of the inhibitor to myosin and the irradiation eliminates the residual activity of unbound inhibitor molecules, azidoblebbistatin has a great potential to become a highly effective tool in both structural studies of actomyosin contractility and the investigation of cellular and physiological functions of myosin II. We used azidoblebbistatin to identify previously unknown low-affinity targets of the inhibitor (EC50 >= 50 mu M) in Dictyostelium discoideum, while the strongest interactant was found to be myosin II (EC50 = 5 mu M). Our results demonstrate that azidoblebbistatin, and potentially other azidated drugs, can become highly useful tools for the identification of strong-and weak-binding cellular targets and the determination of the apparent binding affinities in in vivo conditions.
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