Azidoblebbistatin, a photoreactive myosin inhibitor

Abstract

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 (EC 50 ≥ 50 μM) in Dictyostelium discoideum, while the strongest interactant was found to be myosin II (EC 50 = 5 μM). 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. interactom profile | azidation | photoactivation V arious biological processes, including muscle contraction, cell migration, differentiation, and cytokinesis require the activity of myosin II, a class of actin-based ATP-driven motor proteins. Cell-permeable small molecules that can perturb myosin functions have greatly aided the dissection and understanding of molecular processes underlying the above phenomena. Myosin II inhibitors described to date include 2,3-butanedione monoxime (BDM) (1, 2), N-benzyl-p-toluenesulphonamide (BTS) (3), and blebbistatin (4-6). As BDM has turned out to have a broad effect on many other proteins (7) and the inhibitory effect of BTS is limited to fast skeletal muscle myosin, until now blebbistatin has been the only potent tool for specific blocking of myosin II-dependent processes in various species and cell types. Recent data have shown that halogenated pseudilins also have nonspecific inhibitory effects on myosin II isoforms (8-10).In vitro, blebbistatin inhibits vertebrate striated muscle and nonmuscle myosin II isoforms as well as Dictyostelium discoideum (Dd) myosin II by about 95%, with an IC 50 of 0.5-5 μM (11). Vertebrate smooth muscle and Acanthamoeba myosin II are incompletely inhibited even at high blebbistatin concentrations. In vivo experiments performed with Dd showed that the effective inhibition of myosin II-dependent processes, including growth in...

Various Themes of Myosin Regulation

“…Among those, para-nitroblebbistatin is the most latest described non-cytotoxic and photostable blebbistain derivative that displays the same inhibitory potency to myosins-2 as blebbistatin and allows studies of GFP-nonmuscle myosin-2 inhibition in live cells [204]. Another blebbistatin derivative, photoreactive azidoblebbistatin, covalently crosslinks to the myosin-2 motor domain after UV-irradiation [205]. This photoreactivity improves the in vitro and in vivo effectiveness when compared to blebbistatin but its binding to the myosin-2 motor domain is irreverible [205].…”

“…Another blebbistatin derivative, photoreactive azidoblebbistatin, covalently crosslinks to the myosin-2 motor domain after UV-irradiation [205]. This photoreactivity improves the in vitro and in vivo effectiveness when compared to blebbistatin but its binding to the myosin-2 motor domain is irreverible [205]. …”

para‐Nitroblebbistatin, the Non‐Cytotoxic and Photostable Myosin II Inhibitor

“…[5] For in vivo applications of blebbistatin, optimization of its chemical structure is essential. Introduction of a nitro group at the C7 position (Scheme 1) of the tricyclic core of blebbistatin (7) led to slightly decreased fluorescence and increased photostability. [6] However, the biological application of this blebbistatin derivative was hampered by its strongly decreased binding constant and specificity to myosin II.…”

“…[6] However, the biological application of this blebbistatin derivative was hampered by its strongly decreased binding constant and specificity to myosin II. Recently, we reported a highly myosin II-specific, photoinducible, C15-substituted azido derivative of blebbistatin, called para-azidoblebbistatin, [7] which forms covalent cross-links with its target proteins upon UV and bluelight irradiation. We observed that paraazidoblebbistatin-unlike blebbistatin-is nonfluorescent Scheme 1.…”

“…[8] We synthesized para-nitroblebbistatin (6) by nitration of the pyrrolidinone building block of blebbistatin and subsequent application of Davis oxaziridine protocol [6] (Scheme 1). para-Chloroblebbistatin (8) was synthesized by the direct chlorination of blebbistatin (7).…”