# Crystal structures of Ryanodine Receptor reveal dantrolene and azumolene interactions guiding inhibitor development

## TL;DR

Researchers uncovered how dantrolene and azumolene bind to a key receptor, offering insights to develop better inhibitors.

## Contribution

High-resolution crystal structures of the RyR Repeat12 domain bound to dantrolene, azumolene, and nucleotides are reported for the first time.

## Key findings

- Dantrolene and azumolene bind cooperatively with nucleotides in a pseudosymmetric cleft involving Trp880 and Trp994.
- Binding induces a clamshell-like closure of the R12 domain and allosterically influences RyR gating.
- Structure-based screening identified a potent compound with a distinct binding mode targeting the same site.

## Abstract

The ryanodine receptor (RyR) is a critical drug target, yet dantrolene (DAN) remains the only FDA-approved inhibitor, limited by hepatotoxicity and unsuitable for chronic use. To guide improved inhibitor development, we determine high-resolution crystal structures of the RyR Repeat12 (R12) domain bound to DAN, its analog azumolene (AZU), and adenine nucleotides (AMP-PCP or ADP). DAN/AZU and nucleotides bind cooperatively to a pseudosymmetric cleft, with key interactions involving Trp880 and Trp994. Binding induces a clamshell-like closure of the R12 domain. Isothermal titration calorimetry (ITC) reveals higher affinity in the presence of nucleotides and lower affinity for RyR2 due to nearby substitutions. Structural comparison with cryo-EM data suggests that DAN/AZU binding allosterically influences RyR gating and functional regulation. Structure-based screening identifies a potent compound targeting the same site but with a distinct binding mode. Our findings highlight the power of domain-focused crystallography in guiding RyR inhibitor discovery and overcoming cryo-EM resolution limitations.

This study reports crystal structures of the ryanodine receptor domain bound to dantrolene and azumolene. The work reveals cooperative nucleotide binding and provides structural insights to guide the design of potent inhibitors targeting the receptor.