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

Várkuti, Boglárka H.; Képiró, Miklós; Horváth, I.; Végner, László; Ráti, Szilvia; Zsigmond, Áron; Hegyi, György; Lenkei, Zsolt; Varga, Máté; Málnási‐Csizmadia, András

doi:10.1038/srep26141

Cited by 99 publications

(149 citation statements)

References 26 publications

(50 reference statements)

Supporting

Mentioning

139

Contrasting

Order By: Relevance

“…This state has been referred to as the super relaxed state (SRX) and Bleb has been shown to stabilize the SRX by unknown mechanisms (Wilson et al, 2014). The use of Bleb was hindered by its blue light sensitivity, phototoxicity, and poor solubility (Sakamoto et al, 2005; Mikulich et al, 2012), but this has been addressed by the discoveries of highly soluble, non-phototoxic Bleb derivatives [para-Nitroblebbistatin (pN-Bleb), and amino-blebbistatin; Képiró et al, 2014; Várkuti et al, 2016]. …”

Section: Introductionmentioning

confidence: 99%

Modulating Beta-Cardiac Myosin Function at the Molecular and Tissue Levels

et al. 2017

Self Cite

View full text Add to dashboard Cite

Inherited cardiomyopathies are a common form of heart disease that are caused by mutations in sarcomeric proteins with beta cardiac myosin (MYH7) being one of the most frequently affected genes. Since the discovery of the first cardiomyopathy associated mutation in beta-cardiac myosin, a major goal has been to correlate the in vitro myosin motor properties with the contractile performance of cardiac muscle. There has been substantial progress in developing assays to measure the force and velocity properties of purified cardiac muscle myosin but it is still challenging to correlate results from molecular and tissue-level experiments. Mutations that cause hypertrophic cardiomyopathy are more common than mutations that lead to dilated cardiomyopathy and are also often associated with increased isometric force and hyper-contractility. Therefore, the development of drugs designed to decrease isometric force by reducing the duty ratio (the proportion of time myosin spends bound to actin during its ATPase cycle) has been proposed for the treatment of hypertrophic cardiomyopathy. Para-Nitroblebbistatin is a small molecule drug proposed to decrease the duty ratio of class II myosins. We examined the impact of this drug on human beta cardiac myosin using purified myosin motor assays and studies of permeabilized muscle fiber mechanics. We find that with purified human beta-cardiac myosin para-Nitroblebbistatin slows actin-activated ATPase and in vitro motility without altering the ADP release rate constant. In permeabilized human myocardium, para-Nitroblebbistatin reduces isometric force, power, and calcium sensitivity while not changing shortening velocity or the rate of force development (ktr). Therefore, designing a drug that reduces the myosin duty ratio by inhibiting strong attachment to actin while not changing detachment can cause a reduction in force without changing shortening velocity or relaxation.

show abstract

Section: Introductionmentioning

confidence: 99%

Modulating Beta-Cardiac Myosin Function at the Molecular and Tissue Levels

et al. 2017

Self Cite

View full text Add to dashboard Cite

show abstract

“…HeLa Kyoto cells to visualize the multinucleated phenotype after treating cells with blebbistatin derivatives 8,9 . Because these cells express EGFP-α-tubulin in the cytoplasm and mCherry-H2B in nuclei, they do not require any staining before imaging.…”

Section: Resultsmentioning

confidence: 99%

“…This results in the accumulation of binucleated cells in the population 7 . Para-nitroblebbistatin and para-aminoblebbistatin are photostable derivatives of blebbistatin with reduced cytotoxicity 8,9 . Para-aminoblebbistatin also shows highly improved water solubility 9 .…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

A Simple and Robust Cell-Based Assay for the Discovery of Novel Cytokinesis Inhibitors

Radnai

Stremel

Vaissière

et al. 2020

Preprint

View full text Add to dashboard Cite

Cytokinesis is the last step of mitotic cell division that separates the cytoplasm of dividing cells.Small molecule inhibitors targeting either the elements of the regulatory pathways controlling cytokinesis, or the terminal effectors have been of interest as potential drug candidates for the treatment of various diseases. Here we have developed a cell-based assay for the discovery of novel cytokinesis inhibitors. The assay is performed in a 96-well plate format in 48 hours. Living cells, nuclei and nuclei of dead cells are revealed by a single staining step with three fluorescent dyes, followed by live cell imaging using an automated fluorescence microscope. The primary signal is the nuclei-to-cell ratio (NCR). In the presence of cytokinesis inhibitors, this ratio is expected to increase over time, as the ratio of multinucleated cells increases in the population.Cytotoxicity is quantified as the ratio of dead nuclei to total nuclei. A screening window coefficient (Z`) of 0.65 indicates that the assay is suitable for screening purposes, as the positive and negative controls are well-separated. EC 50 values for four compounds can be reliably determined in a single 96-well plate by using only six different compound concentrations. An excellent test-retest reliability (R 2 =0.998) was found for EC 50 values covering a ~1500-fold range of potencies. The robustness, simplicity and flexibility of the assay is demonstrated here by using modulators of actin dynamics with different mechanisms of action (jasplakinolide, cytochalasin D and swinholide A) and derivatives of the nonmuscle myosin II inhibitor blebbistatin.

show abstract

“…The optimized single molecule ATPase assay enabled us to directly observe effects of the myosin inhibitor para-aminoblebbistatin, a light insensitive, non-fluorescent and highly, soluble blebbistatin analog ( Fig. 5a) (40,41). In this proof of principle drug testing experiment, we used (Fig.…”

Section: Effect Of Para-aminoblebbistatin (Ambleb) On Myosin Basal Atmentioning

confidence: 99%

Optimized single molecule fluorescence sheds light on elusive enzymatic mechanisms

Ušaj

Moretto

Vemula

et al. 2020

Preprint

View full text Add to dashboard Cite

Author ContributionsA.M. and M.U. conceived the project, M.U. and A.M. designed the TIRF system. M.U. and A.M. designed the experiments. M.U. built the TIRF system and performed single molecule fluorescence experiments. M.U. L.M. and A.M. analyzed TIRF data, L.M. performed the bioinformatics modelling. V.V. performed in vitro motility assay experiments with and without AmBleb and analyzed the data, and A.S. analyzed in vitro motility assay data, A.M., M.U., L.M., V.V. and A.S. wrote the manuscript and approved the final version. AbstractSingle molecule enzymology using fluorescent substrate requires truly minimal amounts of proteins. This is highly beneficial when the protein source is either advanced expression systems or samples from humans/animals with ethical and economic implications. Further benefits of single molecule analysis is the potential to reveal phenomena hidden in ensemble studies. However, dye photophysics and fluorescent contaminants complicate interpretation of the single molecule data. We here corroborate the importance of such complexities using fluorescent Alexa647 ATP to study ATP turnover by myosin and actomyosin. We further show that the complexities are largely eliminated by aggressive surface cleaning and use of a range of triple state quenchers and redox agents with minor effects on actin-myosin function. Using optimized assay conditions, we then show that the distributions of ATP binding dwell times on myosin are best described by the sum of 2 to 3 exponential processes. This applies in the presence and absence of actin and in the presence and absence of the drug para-aminoblebbistatin. Two of the processes are attributable to ATP turnover by myosin and actomyosin, respectively. A remaining process with rate constant in the range 0.2-0.5 s -1 is consistent with non-specific ATP binding to myosin and bioinformatics modelling suggests that such binding may be important for accelerated ATP transport to the active site. Finally, we report studies of the actin-activated myosin ATP turnover under conditions with no sliding between actin and myosin, as in isometrically contracting muscle, revealing heterogeneity in the ATP turnover kinetics between different molecules.

show abstract

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

Cited by 99 publications

References 26 publications

Modulating Beta-Cardiac Myosin Function at the Molecular and Tissue Levels

Modulating Beta-Cardiac Myosin Function at the Molecular and Tissue Levels

A Simple and Robust Cell-Based Assay for the Discovery of Novel Cytokinesis Inhibitors

Optimized single molecule fluorescence sheds light on elusive enzymatic mechanisms

Contact Info

Product

Resources

About