Targeting ryanodine receptors with allopurinol and xanthine derivatives for the treatment of cardiac and musculoskeletal weakness disorders

Figures

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  Fig. 1.. Cryo-EM of xanthine derivatives, analogs, and related compounds in the xanthine/caffeine binding site of RyRs. (A) Atomic model of RyR1 (PDB:9E17, gray), highlighting the location of Ca2+ (green), caffeine (red), ATP (dark green), the signal transducing domains CTD (blue) and TaF (pink), and the pore S6 helix (orange). To facilitate visualization, only the front protomer is shown, while the other three protomers are shown as transparent volumes. The sarcoplasmic reticulum membrane is shown as black discs. (B) For each compound, we show the chemical structure (Left) and the sharpen cryo-EM map (gray) overlapped with the respective atomic model (cartoon), local resolution achieved, and PDB identifier (Right). For clarity, the atom indexing is presented only for xanthine (inner numbers). All tested xanthine, allopurinol, and uracil derivatives bind to the xanthine/caffeine site except for xanthosine and uridine.

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  Fig. 2.. Coordination mode of xanthine and oxopyricid. (A and B) Coordination mode of xanthine in the xanthine/caffeine binding site of RyR2, showing pi stacking of xanthine with RyR2–Trp4645 (A), and hydrogen bonding with RyR2–Tyr4944 and RyR2–Glu4194, through a water bridge (B). Measured distances (yellow dashed lines) and angles (black curve lines) suggest a strong hydrogen bond network. (C and D) Coordination mode of oxopyricid in the xanthine/caffeine binding site of RyR1, showing pi stacking of oxopyricid with RyR1–Trp4716 (C), and hydrogen bonding with RyR1–Tyr5014, RyR1–Gln4246, and RyR1–Glu4239, through a water bridge (D). Measured distances (yellow dashed lines) and angles (black curve lines) suggest a strong hydrogen bond network.

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  Fig. 3.. Xanthine derivatives and related compounds increase ryanodine binding and muscle twitch force. (A) Ryanodine binding assay of rabbit RyR1 microsomes in the presence of 1 mM of caffeine, pentoxifylline, dyphylline, oxypurinol, or oxopyricid at increasing Ca2+ free concentrations (50 nM, 150 nM, 500 nM, 1 µM, 3 µM, 10 µM, and 100 µM). For 1 µM and 3 µM Ca2+ free concentrations, all compounds showed significant difference with the control (t test, *P < 0.05, n = 2). For visualization purposes, a sigmoidal curve was fitted for each compound dataset (dotted line). (B–E) EDL twitch force measurement before and after a 5-min incubation with Krebs buffer in the presence of 5 mM of pentoxifylline (B), dyphylline (C), oxypurinol (D), or caffeine (E). Using a paired t test, EDL muscles showed significant increase in twitch force in the presence of pentoxifylline (P = 0.006, mean difference=2.48, n = 5), dyphylline (P = 0.025, mean difference = 1.27, n = 5), oxypurinol (P = 0.013, mean difference = 0.66, n = 5), or caffeine (P = 0.014, mean difference = 2.98, n = 5). (F) EDL twitch force measurement before and after a 5-min incubation with Krebs buffer (wash) of EDL preincubated with…

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  Fig. 4.. Ryanodine receptors activity enhancers as potential therapeutics. (A–C) Schematic representation of RyRs and muscle contraction in healthy conditions (A), in diseases involving loss-of-function RyRs mutations (B), and in diseases involving reduced expression of RyRs (C). (D) Minimal motif necessary for compounds to bind to the xanthine/caffeine site of RyRs and to be used as a scaffold to develop specific RyRs activity enhancers. (E and F) Schematic representation of RyRs and muscle contraction in the presence of RyRs activity enhancers in diseases involving loss-of-function RyRs mutations (E) and in diseases involving reduced expression of RyRs (F).