The type 1 ryanodine receptor (RyR1) is a Ca2+ release channel in skeletal muscle. RyR1 is also expressed in brain and nonmuscle tissues and hyperactivation of RyR1 is implicated not only in skeletal ...muscle diseases, such as malignant hyperthermia, but also in diseases of the brain and other tissues. Therefore, pharmacological inhibition of RyR1 may have a therapeutic potential for various diseases. We have developed Cpd1, a selective RyR1 inhibitor for the treatment of malignant hyperthermia, but it has disadvantages of low oral efficacy and short half-life. To identify novel inhibitors with improved properties, in this study, we aimed to establish in silico screening platform based on the Cpd1-RyR1 complex structure predicted by molecular dynamics simulations. First, we performed amino acid substitutions and Cpd1 modifications on the computer and ran simulations with these modifications. Second, based on these results, amino acid substitution mutants and Cpd1 derivatives were evaluated by biochemical experiments. The results showed that the molecular dynamics simulations were in good agreement with the biochemical experiments. The results of in silico screening with the platform will be presented.
RyR1 is a calcium release channel in the sarcoplasmic reticulum which is involved in excitation-contraction coupling of skeletal muscle. Mutations in RyR1 are known to cause malignant ...hyperthermia. We have recently developed a RyR1 inhibitor, Cpd1, for the treatment of malignant hyperthermia. Although Cpd1 is predicted to bind to the P1 domain of RyR1, the cryo-EM structure of RyR1 complexed with Cpd1 has not been obtained yet, due to low resolution of the P1 domain. Here, we performed molecular dynamics simulations to predict the Cpd1-bound structure of the P1 domain. The simulations were performed using the molecular dynamics program myPresto with different initial coordinates and simulation time. The stability of the output structures was evaluated by the amino acid interactions, and the convergence of the conformational change was evaluated by the RMSD plot. We obtained a stable complex structure of P1 with Cpd1from several initial coordinates with longer simulation time. Interestingly, the structure of P1 complexed with Cpd1 was significantly different from the initial conformation without Cpd1, suggesting that Cpd1 may induce a significant conformational change of the P1 domain.
In skeletal muscle, depolarization of the plasma membrane triggers Ca2+ release from the sarcoplasmic reticulum (SR),referred to as depolarization-induced Ca2+ release (DICR). DICR occurs via the ...type 1 ryanodine receptor (RyR1), which physically interacts with the dihydropyridine receptor Cav1.1 subunit in specific machinery formed with additional essential components including β1a, Stac3 adaptor protein and junctophilins. It has recently become clear that mutations in these components cause various skeletal muscle diseases. However, no specific treatment has been developed yet. In this study, we established a high-throughput platform of the reconstituted DICR in HEK293 cells. The essential components were effectively transduced using baculovirus vectors, and Ca2+ release was quantitatively measured with R-CEPIA1er, a fluorescent ER Ca2+ indicator. High K+ depolarization triggered rapid Ca2+ release, indicating successful reconstitution of DICR. We tested several known drugs modulating DICR. Whereas RyR1 inhibitors, dantrolene and Cpd1, suppressed DICR, twitch potentiators, e.g., perchlorate, accelerated DICR. These results well reproduced the findings with the muscle fibers and the cultured myotubes. The reconstituted DICR platform will be highly useful for drug discovery for skeletal muscle diseases.
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