Improved Inhibitory and Absorption, Distribution, Metabolism, Excretion, and Toxicology (ADMET) Properties of Blebbistatin Derivatives Indicate That Blebbistatin Scaffold Is Ideal for drug Development Targeting Myosin-2

Abstract

Blebbistatin, para-nitroblebbistatin (NBleb) and para-aminoblebbistatin (AmBleb) are highly useful tool compounds as they selectively inhibit the ATPase activity of myosin-2 family proteins. Despite the medical importance of the myosin-2 family as drug targets, chemical optimization has not yet provided a promising lead for drug development because previous structure-activity-relationship (SAR) studies were limited to a single myosin-2 isoform. Here we evaluated the potential of blebbistatin scaffold for drug development and found that Dring substitutions can fine-tune isoform specificity, ADME and toxicological properties. We defined the inhibitory properties of NBleb and AmBleb on seven different myosin-2 isoforms, which revealed an unexpected potential for isoform specific inhibition. We also found that NBleb metabolizes six times slower than blebbistatin and AmBleb in rats, while AmBleb metabolizes two times slower than blebbistatin and NBleb in human, and that AmBleb accumulates in muscle tissues. Moreover, mutagenicity was also greatly reduced in case of AmBleb. These results demonstrate that small substitutions have beneficial functional and pharmacological consequences, which highlight the potential of the blebbistatin scaffold for drug development targeting myosin-2 family proteins and delineate a route for defining the chemical properties of further derivatives to be developed. Significance statement: Small substitutions on the blebbistatin scaffold have beneficial functional and pharmacological consequences highlighting their potential in drug development targeting myosin-2 family proteins.

Pharmacological Modulation of Neurite Outgrowth in Human Neural Progenitor Cells by Inhibiting Non-muscle Myosin II

“…Two hours after seeding eGFP-NPCs onto 96-well plates previously coated with various ECM components (3-5 × 10 3 /well), the cells were subjected to NMII, Rho-associated, coiled-coil-containing protein kinase 1 (ROCK1), or c-Jun N-terminal kinase (JNK) inhibitors at various concentrations, as indicated. While NBS and AmBS were produced as described previously (Gyimesi et al, 2021), the ROCK1-selective inhibitor Y27632 and the pan-JNK inhibitor SP600125 were purchased from Sigma. Green fluorescence images were acquired by an ImageXpress Micro XLS instrument (Molecular Devices) equipped with environment control unit providing 37 • C temperature and humidified atmosphere containing 5% CO 2 , 20% O 2 , and 75% N 2 .…”

“…To overcome these negative properties of blebbistatin, several BS-derivatives have recently been developed. The most promising derivatives, which are non-fluorescent, highly soluble and non-toxic compounds, include para-nitroblebbistatin (NBS) and para-aminoblebbistatin (AmBS) (Kepiro et al, 2014;Varkuti et al, 2016;Rauscher et al, 2018;Gyimesi et al, 2021).…”

Pharmacological Modulation of Neurite Outgrowth in Human Neural Progenitor Cells by Inhibiting Non-Muscle Myosin II

“…After 2 hours, the cells were subjected to NMII, ROCK1, or JNK inhibitors at various concentrations, as indicated. While NBS and AmBS were produced as described previously (33), the ROCK1-selective inhibitor Y27632 and the pan-JNK inhibitor SP600125 were purchased from Sigma. For quantitative analysis, the total neurite lengths were assessed in each eld of view using the Neurite Outgrowth module of MetaXpress software (Molecular Devices).…”

“…To overcome these negative properties of blebbistatin several BS-derivatives have recently been developed. The most promising derivatives, which are non-uorescent, highly soluble and non-toxic compounds, include para-nitroblebbistatin (NBS) and paraaminoblebbistatin (AmBS) (30)(31)(32)(33).…”

Altered contractility in mutation-specific hypertrophic cardiomyopathy: a mechano-energetic in silico study with pharmacological insights

“…MAVA is an allosteric inhibitor of cardiac myosin ATPase with a negative inotropic effect and demonstrating efficacy in R403Q HCM clinical trials [3,10]. BLEB, a well characterized ATPase inhibitor, alters the Ca 2+ sensitivity of the myofilament and has been widely used in trials [11][12][13][14]. OM is a recently developed myosin ATPase activator with a positive inotropic effect enhancing cardiac contractility [15].…”