Abstract
Blebbistatin is a commonly used molecular tool for the specific inhibition of various myosin II isoforms both in vitro and in vivo. Despite its popularity, the use of blebbistatin is hindered by its poor water-solubility (below 10 micromolar in aqueous buffer) and blue-light sensitivity, resulting in the photoconversion of the molecule, causing severe cellular phototoxicity in addition to its cytotoxicity. Furthermore, blebbistatin forms insoluble aggregates in water-based media above 10 micromolar with extremely high fluorescence and also high adherence to different types of surfaces, which biases its experimental usage. Here, we report a highly soluble (440 micromolar in aqueous buffer), non-fluorescent and photostable C15 amino-substituted derivative of blebbistatin, called para-aminoblebbistatin. Importantly, it is neither photo- nor cytotoxic, as demonstrated on HeLa cells and zebrafish embryos. Additionally, para-aminoblebbistatin bears similar myosin II inhibitory properties to blebbistatin or para-nitroblebbistatin (not to be confused with the C7 substituted nitroblebbistatin), tested on rabbit skeletal muscle myosin S1 and on M2 and HeLa cells. Due to its drastically improved solubility and photochemical feature, as well as lack of photo- or cytotoxicity, para-aminoblebbistatin may become a feasible replacement for blebbistatin, especially at applications when high concentrations of the inhibitor or blue light irradiation is required.
Highlights
Blebbistatin[1] is a widely used and well-characterized small molecular inhibitor of class II myosins, a group of ATP driven molecular motors acting in concert with actin to form a contracting actomyosin network
Para-aminoblebbistatin was synthesized by the reduction of para-nitroblebbistatin in the presence of ammonium formate using palladium black catalyst (Fig. 1)
In the majority of the published studies, blebbistatin is applied at 50–100 μM concentrations, at such concentrations blebbistatin precipitates and the effective concentration exponentially drops to the equilibrium saturation concentration (9 μM) in two hours
Summary
Blebbistatin[1] is a widely used and well-characterized small molecular inhibitor of class II myosins, a group of ATP driven molecular motors acting in concert with actin to form a contracting actomyosin network. The application of blebbistatin at higher concentrations than its solubility faces several hurdles in long-term experiments: I) the concentration of the inhibitor decreases over time, and so does its inhibitory effect, II) the light scattering of the media gradually increases, confounding/perturbing light-scattering based measurements, III) the precipitated aggregates have high fluorescence hampering imaging, IV) the aggregates can block the vascular system of animals in in vivo studies[10]. The electron withdrawing nitro substitution at the C15 position diminishes blebbistatin’s cyto- and phototoxicity, reduces its fluorescence and increases its photostability[8] Based on these observations we speculated that substituting a polar, electron withdrawing group at this position may offer the benefits of para-nitroblebbistatin but would elevate the water solubility of the new derivative. Para-aminoblebbistatin forms a stable solution in aqueous buffers and does not precipitate
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