A highly soluble, non-phototoxic, non-fluorescent blebbistatin derivative

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.

Figure 1. Synthesis of para-aminoblebbistatin.

Reagents and conditions: (a) H₂SO₄, HNO₃, 0 °C, 15 min; (b) POCl₃, CH₂Cl₂, 50 °C, 18 hours; (c) LiHMDS, −78 °C to 0 °C, 3 hours; (d) oxaziridine, −10 °C, 16 hours; (e) NH₄HCO₂, Pd black, CH₃OH, RT, 18 hours.

Figure 2. Physico-chemical properties of para-aminoblebbistatin (AmBleb), para-nitroblebistatin (NBleb) and blebbistatin (Bleb).

(a) Solubility of AmBleb, NBleb and Bleb in 0.1 vol/vol% DMSO in assay buffer in time. After the centrifugation of a 500 μM of AmBleb suspension in assay buffer yielded 298 ± 2.5 μM soluble supernatant concentration. The concentration of this solution stayed constant for 4 hours. Supernatant concentrations of 50 μM of NBleb and Bleb decreased exponentially after centrifugation at different lengths of time (enlarged in the inset), reaching equilibria at 3.3 ± 0.1 μM and 10.9 ± 0.9 μM, respectively (obtained from fitting the data to single exponential functions). (b) Solubility of AmBleb, NBleb and Bleb in 1 vol/vol% DMSO are 426 ± 1.7 μM, 3.6 ± 0.2 μM and 9.3 ± 0.7 μM, respectively (enlarged in the inset, obtained from the exponential fits to data). (c) Fluorescence spectra of AmBleb, NBleb and Bleb at λ_exc = 350 nm. (d) Absorbance spectra of AmBleb and Bleb (inset) after irradiating 5 μM of the inhibitors at 480 ± 10 nm for different lengths of time.

Figure 3. In vitro and in vivo inhibitory properties of para-aminoblebbistatin, para-nitroblebbistatin and blebbistatin.

In vitro inhibition of basal (a) and actin activated (b) ATPase activity of rabbit skeletal S1 (SkS1) and Dictyostelium discoideum myosin II motor domain (DdMD) at increasing concentrations of AmBleb, NBleb and Bleb. Data (means ± s.d. from three independent experiments) were normalized to ATPase activities at 0 μM inhibitor concentrations and were fitted with hyperbolic functions. (c–e) Blebbing indices of M2 cells in the presence of increasing concentrations of AmBleb (c), NBleb (d) and Bleb (e) monitored for 60 minutes. Blebbing indices are means and standard deviations/means and s.d./means ± s.d. (n = 6) of the number of blebs formed in a 5-minute interval on a cell. Blebbing index values are normalized to the starting point. Data were fitted with exponentials yielding rate constants of inhibition for 5, 10, 25 and 50 μM of the inhibitors (see Table 1.) (f) Relative migration of HeLa cells measured after 24 h in the absence (Control) or in the presence of 20 μM AmBleb, NBleb and Bleb (left panel). 100% is considered as the total diameter of the wound (n = 6 scratches, from two independent experiments). Representative images of wound healing assays immediately after scratching (0 min) and after 24 h incubation with AmBleb or without the inhibitors (Control). White lines mark the cell edges at 0 minutes (g). Inhibition of cell number growth of HeLa cells by increasing concentrations of AmBleb, NBleb and Bleb monitored for three days. Cell numbers were normalized to 0 μM inhibitor concentrations counted on the first day of the experiment. (h) Cell number as a function of increasing inhibitor concentrations at day 3 of the experiment presented in (g). Data (means ± s.d., n = 50–100) were fitted with hyperboles.

Figure 4. Phototoxicity and cytotoxicity assays with para-aminoblebbistatin or blebbistatin and fluorescent imaging in the presence of the myosin II specific inhibitors.

(a) Maximum intensity projections of fluorescent confocal microscopic z-stack images of EGFP-α-tubulin H2B-mCherry HeLa Kyoto cells at the beginning and at the end of a 12-hour time-lapse imaging in the presence of 50 μM AmBleb or Bleb. Blebbistatin treated cells display severe phototoxicity (white arrowhead). (b) Lifespan cytotoxicity assays of zebrafish embryos (n = 20) incubated at increasing concentrations of AmBleb and Bleb (inset) in dark. (c) Fluorescent confocal microscopic images of HeLa Kyoto cells in the presence of 50 μM inhibitor concentrations on the 3^rd day of the experiment presented in Fig. 3e,f. (d) 48 hpf (hours-post-fertilization) cldnb:EGFP zebrafish embryos in the absence (Control) and in the presence of 20 μM of the myosin II inhibitors after 24 hours of incubation. Red arrowheads mark the position of the pLLps.