The design of compounds that can discriminate between closely related target proteins remains a central challenge in drug discovery. Specific therapeutics targeting the highly conserved myosin motor family are urgently needed as mutations in at least 6 of its members cause numerous diseases. Allosteric modulators, like the myosin-II inhibitor blebbistatin, are a promising means to achieve specificity. However, it remains unclear why blebbistatin inhibits myosin-II motors with different potencies given that it binds at a highly conserved pocket that is always closed in blebbistatin-free experimental structures. We hypothesized that the probability of pocket opening is an important determinant of the potency of compounds like blebbistatin. To test this hypothesis, we used Markov state models (MSMs) built from over 2 milliseconds of aggregate molecular dynamics simulations with explicit solvent. We find that blebbistatin’s binding pocket readily opens in simulations of blebbistatin-sensitive myosin isoforms. Comparing these conformational ensembles reveals that the probability of pocket opening correctly identifies which isoforms are most sensitive to blebbistatin inhibition and that docking against MSMs quantitatively predicts blebbistatin binding affinities (R2=0.82). In a blind prediction for an isoform (Myh7b) whose blebbistatin sensitivity was unknown, we find good agreement between predicted and measured IC50s (0.67 mM vs. 0.36 mM). Therefore, we expect this framework to be useful for the development of novel specific drugs across numerous protein targets.
Experimental, pocket volume, docking, and trajectory clustering data have been deposited in OSF under accession code CV6D2. Scripts and notebooks used to generate all figures are available in our GitHub repository (https://github.com/bowman-lab/blebbistatin-specificity).
- Artur Meller
- Gregory R Bowman
- Gregory R Bowman
- Michael J Greenberg
- Leslie A Leinwand
- Jeffrey M. Lotthammer
- Gregory R Bowman
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
- Donald Hamelberg, Georgia State University, United States
© 2023, Meller et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
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